Merlin/ERM proteins regulate growth factor-induced macropinocytosis and receptor recycling by organizing the plasma membrane:cytoskeleton interface.

The architectural and biochemical features of the plasma membrane are governed by its intimate association with the underlying cortical cytoskeleton. The neurofibromatosis type 2 (NF2) tumor suppressor merlin and closely related membrane:cytoskeleton-linking protein ezrin organize the membrane:cytoskeleton interface, a critical cellular compartment that both regulates and is regulated by growth factor receptors. An example of this poorly understood interrelationship is macropinocytosis, an ancient process of nutrient uptake and membrane remodeling that can both be triggered by growth factors and manage receptor availability. We show that merlin deficiency primes the membrane:cytoskeleton interface for epidermal growth factor (EGF)-induced macropinocytosis via a mechanism involving increased cortical ezrin, altered actomyosin, and stabilized cholesterol-rich membranes. These changes profoundly alter EGF receptor (EGFR) trafficking in merlin-deficient cells, favoring increased membrane levels of its heterodimerization partner, ErbB2; clathrin-independent internalization; and recycling. Our work suggests that, unlike Ras transformed cells, merlin-deficient cells do not depend on macropinocytic protein scavenging and instead exploit macropinocytosis for receptor recycling. Finally, we provide evidence that the macropinocytic proficiency of NF2-deficient cells can be used for therapeutic uptake. This work provides new insight into fundamental mechanisms of macropinocytic uptake and processing and suggests new ways to interfere with or exploit macropinocytosis in NF2 mutant and other tumors.

Complexities in the role of acetylation dynamics in modifying inducible gene activation parameters.

High levels of histone acetylation are associated with the regulatory elements of active genes, suggesting a link between acetylation and gene activation. We revisited this model, in the context of EGF-inducible gene expression and found that rather than a simple unifying model, there are two broad classes of genes; one in which high lysine acetylation activity is required for efficient gene activation, and a second group where the opposite occurs and high acetylation activity is inhibitory. We examined the latter class in more detail using EGR2 as a model gene and found that lysine acetylation levels are critical for several activation parameters, including the timing of expression onset, and overall amplitudes of the transcriptional response. In contrast, DUSP1 responds in the canonical manner and its transcriptional activity is promoted by acetylation. Single cell approaches demonstrate heterogenous activation kinetics of a given gene in response to EGF stimulation. Acetylation levels modify these heterogenous patterns and influence both allele activation frequencies and overall expression profile parameters. Our data therefore point to a complex interplay between acetylation equilibria and target gene induction where acetylation level thresholds are an important determinant of transcriptional induction dynamics that are sensed in a gene-specific manner.

Integrator regulates transcriptional initiation and pause release following activation.

In unicellular organisms, initiation is the rate-limiting step in transcription; in metazoan organisms, the transition from initiation to productive elongation is also important. Here, we show that the RNA polymerase II (RNAPII)-associated multiprotein complex, Integrator, plays a critical role in both initiation and the release of paused RNAPII at immediate early genes (IEGs) following transcriptional activation by epidermal growth factor (EGF) in human cells. Integrator is recruited to the IEGs in a signal-dependent manner and is required to engage and recruit the super elongation complex (SEC) to EGF-responsive genes to allow release of paused RNAPII and productive transcription elongation.

Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling.

Raf kinases are essential for normal Ras-Raf-MEK-ERK pathway signaling, and activating mutations in components of this pathway are associated with a variety of human cancers, as well as the related developmental disorders Noonan, LEOPARD, and cardiofaciocutaneous syndromes. Although the Raf kinases are known to dimerize during normal and disease-associated Raf signaling, the functional significance of Raf dimerization has not been fully elucidated. Here, using mutational analysis and a peptide inhibitor, we show that dimerization is required for normal Ras-dependent Raf activation and for the biological function of disease-associated Raf mutants with moderate, low, or impaired kinase activity. However, dimerization is not needed for the function of B-Raf mutants with high catalytic activity, such as V600E-B-Raf. Importantly, we find that a dimer interface peptide can effectively block Raf dimerization and inhibit Raf signaling when dimerization is required for Raf function, thus identifying the Raf dimer interface as a therapeutic target.

Frequency-modulated pulses of ERK activity transmit quantitative proliferation signals.

The EGF-stimulated ERK/MAPK pathway is a key conduit for cellular proliferation signals and a therapeutic target in many cancers. Here, we characterize two central quantitative aspects of this pathway: the mechanism by which signal strength is encoded and the response curve relating signal output to proliferation. Under steady-state conditions, we find that ERK is activated in discrete, asynchronous pulses with frequency and duration determined by extracellular concentrations of EGF spanning the physiological range. In genetically identical sister cells, cell-to-cell variability in pulse dynamics influences the decision to enter S phase. While targeted inhibition of EGFR reduces the frequency of ERK activity pulses, inhibition of MEK reduces their amplitude. Continuous response curves measured in multiple cell lines reveal that proliferation is effectively silenced only when ERK pathway output falls below a threshold of ~10%, indicating that high-dose targeting of the pathway is necessary to achieve therapeutic efficacy.

Epidermal growth factor and three-dimensional scaffolds provide conducive environment for differentiation of mouse embryonic stem cells into oocyte-like cells.

Three-dimensional (3D) culture provides a biomimicry of the naive microenvironment that can support cell proliferation, differentiation, and regeneration. Some growth factors, such as epidermal growth factor (EGF), facilitate normal meiosis during oocyte maturation in vivo. In this study, a scaffold-based 3D coculture system using purified alginate was applied to induce oocyte differentiation from mouse embryonic stem cells (mESCs). mESCs were induced to differentiate into oocyte-like cells using embryoid body protocol in the two-dimensional or 3D microenvironment in vitro. To increase the efficiency of the oocyte-like cell differentiation from mESCs, we employed a coculture system using ovarian granulosa cells in the presence or absence of epidermal growth factor (+EGF or -EGF) for 14 days and then the cells were assessed for germ cell differentiation, meiotic progression, and oocyte maturation markers. The cultures exposed to EGF in the alginate-based 3D microenvironment showed the highest level of premeiotic (Oct4 and Mvh), meiotic (Scp1, Scp3, Stra8, and Rec8), and oocyte maturation (Gdf9, Cx37, and Zp2) marker genes (p < .05) in comparison to other groups. According to the gene-expression patterns, we can conclude that alginate-based 3D coculture system provided a highly efficient protocol for oocyte-like cell differentiation from mESCs. The data showed that this culture system along with EGF improved the rate of in vitro oocyte-like cell differentiation.

The Akt-SRPK-SR axis constitutes a major pathway in transducing EGF signaling to regulate alternative splicing in the nucleus.

Pre-mRNA splicing is regulated by developmental and environmental cues, but little is known about how specific signals are transduced in mammalian cells to regulate this critical gene expression step. Here, we report massive reprogramming of alternative splicing in response to EGF signaling. By blocking individual branches in EGF signaling, we found that Akt activation plays a major role, while other branches, such as the JAK/STAT and ERK pathways, make minor contributions to EGF-induced splicing. Activated Akt next branches to SR protein-specific kinases, rather than mTOR, by inducing SRPK autophosphorylation that switches the splicing kinases from Hsp70- to Hsp90-containing complexes. This leads to enhanced SRPK nuclear translocation and SR protein phosphorylation. These findings reveal a major signal transduction pathway for regulated splicing and place SRPKs in a central position in the pathway, consistent with their reputed roles in a large number of human cancers.

The EGF/Ras pathway controls growth in Drosophila via ribosomal RNA synthesis.

The Ras small G-protein is a conserved regulator of cell and tissue growth during animal development. Studies in Drosophila have shown how Ras can stimulate a RAF-MEK-ERK signalling pathway to control cell growth and proliferation in response to Epidermal Growth Factor (EGF) stimulation. This work has also defined several transcription factors that can function as downstream growth effectors of the EGF/Ras/ERK pathway by stimulating mRNA transcription. Here we report on stimulation of RNA polymerase I (Pol I)-mediated ribosomal RNA (rRNA) synthesis as a growth effector of Ras/ERK signalling in Drosophila. We show that Ras/ERK signalling promotes an increase in nucleolar size in larval wing discs, which is indicative of increased ribosome synthesis. We also find that activation of Ras/ERK signalling promotes rRNA synthesis both in vivo and in cultured Drosophila S2 cells. We show that Ras signalling can regulate the expression of the Pol I transcription factor TIF-IA, and that this regulation requires dMyc. Finally, we find that TIF-IA-mediated rRNA synthesis is required for Ras/ERK signalling to drive proliferation in both larval and adult Drosophila tissues. These findings indicate that Ras signalling can promote ribosome synthesis in Drosophila, and that this is one mechanism that contributes to the growth effects of the Ras signalling pathway.

MEF2C regulates outflow tract alignment and transcriptional control of Tdgf1.

Congenital heart defects are the most common birth defects in humans, and those that affect the proper alignment of the outflow tracts and septation of the ventricles are a highly significant cause of morbidity and mortality in infants. A late differentiating population of cardiac progenitors, referred to as the anterior second heart field (AHF), gives rise to the outflow tract and the majority of the right ventricle and provides an embryological context for understanding cardiac outflow tract alignment and membranous ventricular septal defects. However, the transcriptional pathways controlling AHF development and their roles in congenital heart defects remain incompletely elucidated. Here, we inactivated the gene encoding the transcription factor MEF2C in the AHF in mice. Loss of Mef2c function in the AHF results in a spectrum of outflow tract alignment defects ranging from overriding aorta to double-outlet right ventricle and dextro-transposition of the great arteries. We identify Tdgf1, which encodes a Nodal co-receptor (also known as Cripto), as a direct transcriptional target of MEF2C in the outflow tract via an AHF-restricted Tdgf1 enhancer. Importantly, both the MEF2C and TDGF1 genes are associated with congenital heart defects in humans. Thus, these studies establish a direct transcriptional pathway between the core cardiac transcription factor MEF2C and the human congenital heart disease gene TDGF1. Moreover, we found a range of outflow tract alignment defects resulting from a single genetic lesion, supporting the idea that AHF-derived outflow tract alignment defects may constitute an embryological spectrum rather than distinct anomalies.

Two phases of mitogenic signaling unveil roles for p53 and EGR1 in elimination of inconsistent growth signals.

Normal cells require continuous exposure to growth factors in order to cross a restriction point and commit to cell-cycle progression. This can be replaced by two short, appropriately spaced pulses of growth factors, where the first pulse primes a process, which is completed by the second pulse, and enables restriction point crossing. Through integration of comprehensive proteomic and transcriptomic analyses of each pulse, we identified three processes that regulate restriction point crossing: (1) The first pulse induces essential metabolic enzymes and activates p53-dependent restraining processes. (2) The second pulse eliminates, via the PI3K/AKT pathway, the suppressive action of p53, as well as (3) sets an ERK-EGR1 threshold mechanism, which digitizes graded external signals into an all-or-none decision obligatory for S phase entry. Together, our findings uncover two gating mechanisms, which ensure that cells ignore fortuitous growth factors and undergo proliferation only in response to consistent mitogenic signals.

[The role of EGF-like factor signaling pathway in granulosa cells in regulation of oocyte maturation and development].

The surge of luteinizing hormone (LH) in preovulatory ovarian follicles triggers the resumption of meiosis in oocytes and induces the proliferation of surrounding cumulus granulosa cells. It is believed that LH receptors are expressed in the mural granulosa cells, but not the oocytes and the surrounding cumulus cells, suggesting that the LH signaling is mediated by factors produced by the granulosa cells. However, the mechanism underlying oocyte maturation induced by LH before ovulation has been controversial. Current studies suggest that LH binds on to its receptor on granulosa cells of the follicular wall to promote the production of EGF-like factors, which activate various signaling cascades and induce oocyte maturation and development. Since the in vitro maturation system is difficult to simulate the in vivo physiological environment, in vitro cultured follicles are likely to be de?cient in the EGF-like factors, which could result in the poor developmental competency of in vitro cultured oocytes and restrict their efficient utilization. In this review, we summarize the EGF-like factor signaling system in granulosa cells and its regulation of oocyte maturation and development. It aims to optimize the in vitro maturation culture system of oocytes and increase the EGF-like factor signaling system in cumulus granulosa cells, thereby providing a framework for improving the efficiency on in vitro maturation of oocytes.

Colorectal cancer spheroid biobanks: multi-level approaches to drug sensitivity studies.

Biobanking of molecularly characterized colorectal cancer stem cells (CSCs) generated from individual patients and growing as spheroids in defined serum-free media offer a fast, feasible, and multi-level approach for the screening of targeted therapies and drug resistance molecular studies. By combining in vitro and in vivo analyses of cetuximab efficacy with genetic data on an ongoing collection of stem cell-enriched spheroids, we describe the identification and preliminary characterization of microsatellite stable (MSS) CSCs that, despite the presence of the KRAS (G12D) mutation, display epidermal growth factor (EGF)-dependent growth and are strongly inhibited by anti-EGF-receptor (EGFR) treatment. In parallel, we detected an increased resistance to anti-EGFR therapy of microsatellite instable (MSI) CSC lines irrespective of KRAS mutational status. MSI CSC lines carried mutations in genes coding for proteins with a role in RAS and calcium signaling, highlighting the role of a genomically unstable context in determining anti-EGFR resistance. Altogether, these results argue for a multifactorial origin of anti-EGFR resistance that emerges as the effect of multiple events targeting direct and indirect regulators of the EGFR pathway. An improved understanding of key molecular determinants of sensitivity/resistance to EGFR inhibition will be instrumental to optimize the clinical efficacy of anti-EGFR agents, representing a further step towards personalized treatments.

Depletion of Gut Microbiota Protects against Renal Ischemia-Reperfusion Injury.

An accumulating body of evidence shows that gut microbiota fulfill an important role in health and disease by modulating local and systemic immunity. The importance of the microbiome in the development of kidney disease, however, is largely unknown. To study this concept, we depleted gut microbiota with broad-spectrum antibiotics and performed renal ischemia-reperfusion (I/R) injury in mice. Depletion of the microbiota significantly attenuated renal damage, dysfunction, and remote organ injury and maintained tubular integrity after renal I/R injury. Gut flora-depleted mice expressed lower levels of F4/80 and chemokine receptors CX3CR1 and CCR2 in the F4/80+ renal resident macrophage population and bone marrow (BM) monocytes than did control mice. Additionally, compared with control BM monocytes, BM monocytes from gut flora-depleted mice had decreased migratory capacity toward CX3CL1 and CCL2 ligands. To study whether these effects were driven by depletion of the microbiota, we performed fecal transplants in antibiotic-treated mice and found that transplant of fecal material from an untreated mouse abolished the protective effect of microbiota depletion upon renal I/R injury. In conclusion, we show that depletion of gut microbiota profoundly protects against renal I/R injury by reducing maturation status of F4/80+ renal resident macrophages and BM monocytes. Therefore, dampening the inflammatory response by targeting microbiota-derived mediators might be a promising therapy against I/R injury.

Opposing roles of TGF-ß and EGF in the regulation of TRAIL-induced apoptosis in human breast epithelial cells.

Transforming growth factor-beta (TGF-ß) induces the epithelial to mesenchymal transition (EMT) in breast epithelial cells and plays an important role in mammary morphogenesis and breast cancer. In non-transformed breast epithelial cells TGF-ß antagonizes epidermal growth factor (EGF) action and induces growth inhibition. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to participate in lumen formation during morphogenesis of human breast epithelial cells. Our previous work indicated that sensitivity of human breast epithelial cells to TRAIL can be modulated through the activation of the epidermal growth factor receptor-1 (EGFR). Here, we show that TGF-ß opposes EGF-mediated sensitization to TRAIL-induced caspase-8 activation and apoptosis in non-transformed breast epithelial cells. Death-inducing signalling complex (DISC) formation by TRAIL was significantly reduced in cells treated with TGF-ß. TGF-ß treatment activates cytoprotective autophagy and down-regulates TRAIL-R2 expression at the cell surface by promoting the intracellular accumulation of this receptor. Lastly, we demonstrate that EMT is not involved in the inhibitory effect of TGF-ß on apoptosis by TRAIL. Together, the data reveal a fine regulation by EGF and TGF-ß of sensitivity of human breast epithelial cells to TRAIL which may be relevant during morphogenesis.

EGF domain of coagulation factor IX is conducive to exposure of phosphatidylserine.

Lipid rafts are an initiation site for many different signals. Recently, we reported that an EGF domain in activated coagulation factor IX (EGF-F9) increases lipid raft formation and accelerates cell migration. However, the detailed mechanism is not well understood. This study aimed to evaluate the effects of EGF-F9 on the cell membrane. A431 cells (derived from human squamous cell carcinoma) were treated with recombinant EGF-F9. Cells were immunocytochemically stained with probes for lipid rafts or phosphatidylserine (PS). After 3 min of treatment with EGF-F9, cholera toxin subunit B (CTxB) binding domains emerged at the adhesive tips of filopodia. Subsequently, CTxB staining was observed on the filopodial shaft. Finally, large clusters of CTxB domains were observed at the edge of cell bodies. Markers for lipid rafts, such as caveolin-1 and a GPI anchored protein, co-localized with CTxB. Staining with annexin V and XII revealed that PS was exposed at the tips of filopodia, translocated on filopodial shafts, and co-localized with CTxB at the rafts. Immunocytochemistry showed that scramblase-1 protein was present at the filopodial tips. Our data indicates that EGF-F9 accelerates PS exposure around the filopodial adhesion complex and induces clustering of lipid rafts in the cell body. PS exposure is thought to occur on cells undergoing apoptosis. Further study of the function of the EGF-F9 motif in mediating signal transduction is necessary because it is shared by a number of proteins.

A microfluidic Transwell to study chemotaxis.

Chemotaxis is typically studied in vitro using commercially available products such as the Transwell® in which cells migrate through a porous membrane in response to one or more clearly defined chemotactic stimuli. Despite its widespread use, the Transwell assay suffers from being largely an endpoint assay, with built-in errors due to inconsistent pore size and human sampling. In this study, we report a microfluidic chemotactic chip that provides real-time monitoring, consistent paths for cell migration, and easy on-chip staining for quantifying migration. To compare its performance with that of a traditional Transwell chamber, we investigate the chemotactic response of MDA-MB-231 1833 metastatic breast cancer cells to epidermal growth factor (EGF). The results show that while both platforms were able to detect a chemotactic response, we observed a dose-dependent response of breast cancer cells towards EGF with low non-specific migration using the microfluidic platform, whereas we observed a dose-independent response of breast cancer cells towards EGF with high levels of non-specific migration using the commercially available Transwell.The microfluidic platform also allowed EGF-dependent chemotactic responses to be observed 24h, a substantially longer window than seen with the Transwell. Thus the performance of our microfluidic platform revealed phenomena that were not detected in the Transwell under the conditions tested.

Dipeptidyl peptidase 9 enzymatic activity influences the expression of neonatal metabolic genes.

The success of dipeptidyl peptidase 4 (DPP4) inhibition as a type 2 diabetes therapy has encouraged deeper examination of the post-proline DPP enzymes. DPP9 has been implicated in immunoregulation, disease pathogenesis and metabolism. The DPP9 enzyme-inactive (Dpp9 gene knock-in; Dpp9 gki) mouse displays neonatal lethality, suggesting that DPP9 enzyme activity is essential in neonatal development. Here we present gene expression patterns in these Dpp9 gki neonatal mice. Taqman PCR arrays and sequential qPCR assays on neonatal liver and gut revealed differential expression of genes involved in cell growth, innate immunity and metabolic pathways including long-chain-fatty-acid uptake and esterification, long-chain fatty acyl-CoA binding, trafficking and transport into mitochondria, lipoprotein metabolism, adipokine transport and gluconeogenesis in the Dpp9 gki mice compared to wild type. In a liver cell line, Dpp9 knockdown increased AMP-activated protein kinase phosphorylation, which suggests a potential mechanism. DPP9 protein levels in liver cells were altered by treatment with EGF, HGF, insulin or palmitate, suggesting potential natural DPP9 regulators. These gene expression analyses of a mouse strain deficient in DPP9 enzyme activity show, for the first time, that DPP9 enzyme activity regulates metabolic pathways in neonatal liver and gut.

Stem cell factor promotes in vitro ovarian follicle development in the domestic cat by upregulating c-kit mRNA expression and stimulating the phosphatidylinositol 3-kinase/AKT pathway.

In the present study we examined the effects of stem cell factor (SCF; 50 vs 100ngmL-1) alone or in combination with epidermal growth factor (EGF; 100ngmL-1) on: (1) the in vitro viability and growth of cat follicles within ovarian cortices; (2) phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) phosphorylation; and (3) c-kit and FSH receptor (FSHr) mRNA expression. At 100ngmL-1, SCF increased (P≤0.05) the percentage and size of secondary follicles after 14 days of in vitro culture and sustained AKT phosphorylation after 3 days incubation. EGF suppressed this beneficial effect and reduced (P≤0.05) the percentage of structurally normal follicles and FSHr expression when combined with 100ngmL-1 SCF. Expression of c-kit mRNA was higher (P≤0.05) in the presence of 100ngmL-1 SCF compared with fresh follicles and cohorts cultured under other conditions. A c-kit inhibitor suppressed follicle growth and reduced AKT phosphorylation. Collectively, the results demonstrate that SCF promotes cat follicle development by upregulating c-kit mRNA expression and AKT phosphorylation. EGF suppresses the stimulating effect of SCF, leading to downregulation of FSHr expression.

Novel EGFR inhibitors attenuate cardiac hypertrophy induced by angiotensin II.

Cardiac hypertrophy is an important risk factor for heart failure. Epidermal growth factor receptor (EGFR) has been found to play a role in the pathogenesis of various cardiovascular diseases. The aim of this current study was to examine the role of EGFR in angiotensin II (Ang II)-induced cardiac hypertrophy and identify the underlying molecular mechanisms. In this study, we observed that both Ang II and EGF could increase the phospohorylation of EGFR and protein kinase B (AKT)/extracellular signal-regulated kinase (ERK), and then induce cell hypertrophy in H9c2 cells. Both pharmacological inhibitors and genetic silencing significantly reduced Ang II-induced EGFR signalling pathway activation, hypertrophic marker overexpression, and cell hypertrophy. In addition, our results showed that Ang II-induced EGFR activation is mediated by c-Src phosphorylation. In vivo, Ang II treatment significantly led to cardiac remodelling including cardiac hypertrophy, disorganization and fibrosis, accompanied by the activation of EGFR signalling pathway in the heart tissues, while all these molecular and pathological alterations were attenuated by the oral administration with EGFR inhibitors. In conclusion, the c-Src-dependent EGFR activation may play an important role in Ang II-induced cardiac hypertrophy, and inhibition of EGFR by specific molecules may be an effective strategy for the treatment of Ang II-associated cardiac diseases.

The non-receptor tyrosine kinase Ack1 regulates the fate of activated EGFR by inducing trafficking to the p62/NBR1 pre-autophagosome.

Growth factor signalling regulates multiple cellular functions and its misregulation has been linked to the development and progression of cancer. Ack1 (activated Cdc42-associated kinase 1, also known as TNK2) is a non-receptor tyrosine kinase that has been implicated in trafficking and degradation of epidermal growth factor receptor (EGFR), yet its precise functions remain elusive. In this report, we investigate the role of Ack1 in EGFR trafficking and show that Ack1 partially colocalises to Atg16L-positive structures upon stimulation with EGF. These structures are proposed to be the isolation membranes that arise during formation of autophagosomes. In addition, we find that Ack1 colocalises and interacts with sequestosome 1 (p62/SQSTM1), a receptor for selective autophagy, through a ubiquitin-associated domain, and this interaction decreases upon treatment with EGF, thus suggesting that Ack1 moves away from p62/SQSTM1 compartments. Furthermore, Ack1 interacts and colocalises with NBR1, another autophagic receptor, and this colocalisation is enhanced in the presence of ectopically expressed p62/SQSTM1. Finally, knockdown of Ack1 results in accelerated localisation of EGFR to lysosomes upon treatment with EGF. Structure-function analyses of a panel of Ack1 deletion mutants revealed key mechanistic aspects of these relationships. The Mig6-homology domain and clathrin-binding domain both contribute to colocalisation with EGFR, whereas the UBA domain is essential for colocalisation with p62/SQSTM1, but not NBR1. Taken together, our studies demonstrate a novel role for Ack1 in diverting activated EGFR into a non-canonical degradative pathway, marked by association with p62/SQSTM1, NBR1 and Atg16L.