Cell Signaling Systems Research Articles

The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans

Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.

Nitric oxide and cyclic guanosine monophosphate signaling in the eye

This brief review describes the components and pathways utilized in nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) signaling. Since the discovery of the effects of NO and cGMP on smooth muscle relaxation about 30 years ago, the field has expanded in many directions such that many, but not all, biochemical and biological effects seem to be regulated by these unique signaling molecules.While many of the effects of NO are due to activation of soluble guanylyl cyclase (sGC) that can be considered the receptor for NO, cGMP, in turn, can activate a cGMP-dependent protein kinase (PKG) to phosphorylate an array of proteins. Some of the effects of cGMP can be independent of PKG and are due to effects on ion channels or cyclic nucleotide phosphodiesterases. Also, some of the effects of NO can be independent of sGC activation. The isoenzymes and macromolecules that participate in these signaling pathways can serve as molecular targets to identify compounds that increase or decrease their activation and thus serve as chemical leads for discovering novel drugs for a variety of diseases. Some examples are given. However, with about 90,000 publications in the field since our first reports in 1977, this brief review can only give the readers a sample of the excitement and opportunities we have found in this cell signaling system.

Protein Kinase C-ζ is Critical in Pancreatitis-Induced Apoptosis of Kupffer Cells

Protein kinase C-zeta (PKC-ζ) regulates cell death via NF-κB; therefore, we tested the hypothesis that PKC-ζ plays a critical role in pancreatitis-induced Kupffer cell apoptosis. Acute pancreatitis was induced in rats by cerulein injection 24 h later, livers were assayed for PKC-ζ, IKKα, IKKβ, IKKγ, NF-κB, Fas/FasL, and apoptosis was assessed with Caspase-3 and DNA fragmentation. Kupffer cells from unoperated rats were infected with a PKC-ζ domain-negative adenovirus (AdPKCζ-DN) to inhibit PKC-ζ, or transfected with pCMVPKC-ζ to overexpress PKC-ζ, and then stimulated with pancreatic elastase; cellular extracts were assayed for PKC-ζ, IKKα, IKKβ, IKKγ, NF-κB, Fas/FasL, Caspase-3, and DNA fragmentation. Cerulein-induced pancreatitis upregulated PKC-ζ protein and activity, IKKβ, IKKγ, NF-κB, Fas/FasL, Caspase-3 and increased DNA fragmentation in rat livers (all p < 0.001 vs control). AdPKCζ-DN abolished elastase-induced upregulation of PKC-ζ activity, IKKβ, IKKγ, NF-κB, Fas/FasL, Caspase-3 and DNA fragmentation (all p < 0.001 vs infection control), whereas overexpression of PKC-ζ augmented elastase-induced upregulation of IKKβ, IKKγ, Fas/FasL, Caspase-3 and DNA fragmentation (p < 0.001 vs control). PKC-ζ plays a critical role in pancreatitis-induced Kupffer cell apoptosis via NF-κB and Fas/FasL. The ability of Kupffer cells to autoregulate their stress response by upregulating their death receptor/ligand and key proapoptotic cell signaling systems warrants further investigation.

BCR Signaling Generates NF-kB2 (p100) For BR3-Mediated Processing And Survival (83.2)

Abstract Signaling through both the B cell receptor (BCR) and the BLyS receptor, BR3, are required for primary B cell survival. Therefore, understanding how the downstream signaling pathways from these receptors interact should help reveal the molecular mechanisms underlying primary B cell selection and survival. The classical and non-classical NF-kB pathways are an attractive starting point for interrogation, since its members operate downstream of several key B cell signaling systems. Accordingly we have examined BLyS-mediated signaling and survival both in isolation as well as in the context of signals derived form the BCR. In NH3T3 cells expressing BR3, as well as in primary splenic B cells, we find that BLyS signaling via BR3 predominantly activates the non-classical pathway. This is evidenced by an increase in p52 levels and the corresponding diminution of the p100 pool upon BLyS treatment. In contrast, while BCR signaling potently upregulates p100 levels, it does not result in processing to the active p52 form. However, simultaneous BCR and BR3 signaling yields both p52 accumulation and sustained p100 availability. These findings suggest a model whereby BCR signaling is required to maintain available substrate for the viability-promoting BR3 pathway. In support of this model, blockade of proximal BCR signaling with a Syk kinase inhibitor leads to the depletion of p100 stores and the failure of BLyS-mediated survival. Together, these findings lead us to propose a model whereby BCR signaling rations a key survival pathway substrate, thus governing selection and homeostasis.

Fluorescence imaging of cell signaling systems

Fluorescence imaging of cell signaling systems

Cell–cell communication in the plant pathogen Agrobacterium tumefaciens

The plant pathogen Agrobacterium tumefaciens induces the formation of crown gall tumours at wound sites on host plants by directly transforming plant cells. This disease strategy benefits the bacteria as the infected plant tissue produces novel nutrients, called opines, that the colonizing bacteria can use as nutrients. Almost all of the genes that are required for virulence, and all of the opine uptake and utilization genes, are carried on large tumour-inducing (Ti) plasmids. The observation more than 25 years ago that specific opines are required for Ti plasmid conjugal transfer led to the discovery of a cell-cell signalling system on these plasmids that is similar to the LuxR-LuxI system first described in Vibrio fischeri. All Ti plasmids that have been described to date carry a functional LuxI-type N-acylhomoserine lactone synthase (TraI), and a LuxR-type signal receptor and transcriptional regulator called TraR. The traR genes are expressed only in the presence of specific opines called conjugal opines. The TraR-TraI system provides an important model for LuxR-LuxI-type systems, especially those found in the agriculturally important Rhizobiaceae family. In this review, we discuss current advances in the biochemistry and structural biology of the TraR-TraI system.

Evidence for non-chemical, non-electrical intercellular signaling in intestinal epithelial cells

Synchrony between mechanically separated biological systems is well known. We posed the question: can cells induce synchronous behavior in neighboring cells which are mechanically separated and which cannot communicate via chemical or electrical mechanisms. Caco-2 cell cultures were divided into three groups. “Inducer” cells were exposed to H 2O 2. “Detector” cells were placed in separate containers near the inducer cells but were not exposed to H 2O 2. Control cells were exposed to fresh media and were kept in a distant laboratory area. Samples were measured for total protein concentration, NFκB activation and structural changes, 10, 30 and 60 min after exposure respectively. Exposing inducer cells to H 2O 2 resulted in a significant reduction in total protein content (− 50%), an increase in nuclear NFκB activation (+ 38%), and structural damage (56%) compared to controls. There was a similar reduction in total protein content (− 48%), increase in the nuclear fraction of NFκB (+ 35%) and structural damage (25%) in detector cells. These findings provide evidence in support of a non-chemical, non-electrical communication. This signaling system possibly plays a role in synchronous, stimulus-appropriate cell responses to noxious stimuli and may explain a number of cellular behaviors that are hard to explain based only on conventional cell signaling systems.

Association of endometrial cancer risk with a functional polymorphism (Asp327Asn) in the sex hormone-binding globulin gene

Sex steroid hormones play a central role in the development of endometrial cancer. Sex hormone-binding globulin (SHBG) modulates the bioavailability of circulating sex hormones and regulates their signaling systems in target cells. Epidemiologic studies have demonstrated that blood SHBG levels are associated inversely with the risk of endometrial cancer. A functional polymorphism, an amino acid substitution of asparagine (Asp) for aspartic acid (Asn) at residue 327 (Asp(327)Asn) (reference sequence 6259), in the SHBG gene recently was identified and has been associated with an increased half-life and elevated blood levels of SHBG. The authors tested the hypothesis that this genetic variance is associated with a reduced risk of endometrial cancer in the Shanghai Endometrial Cancer Study, a population-based, case-control study that was conducted in urban Shanghai, China between 1997 and 2003. This study included 1037 women with newly diagnosed endometrial cancer ages 30 years and 69 years and 1031 age-matched controls from the community who had completed an in-person interview and donated a blood and/or buccal cell sample to the study. Genotyping for the Asp(327)Asn polymorphism was performed by using the TaqMan method. Odds ratio (ORs) and 95% confidence intervals (95% CIs) derived from logistic regression models were used to evaluate the association between the genetic variation and endometrial cancer risk. The allele frequencies of Asn were 15.0% in cases and 17.1% in controls (P = .06). The variant Asn(327) allele was associated with a reduced risk of endometrial cancer in postmenopausal women (OR, 0.72; 95% CI, 0.55-0.93) but not in premenopausal women (OR, 1.02; 95% CI, 0.74-1.42). The inverse association was more pronounced among postmenopausal women who had a low body mass index (OR, 0.48; 95% CI, 0.31-0.76) or longer years of menstruation (OR, 0.64; 95% CI, 0.46-0.89), although the results from tests for multiplicative interaction were not statistically significant. The current results suggested that the codon 327 Asn allele in the SHBG gene may be related to a reduced risk of endometrial cancer among postmenopausal women.

Comparative developmental neurotoxicity of organophosphates in vivo: Transcriptional responses of pathways for brain cell development, cell signaling, cytotoxicity and neurotransmitter systems

Organophosphates affect mammalian brain development through a variety of mechanisms beyond their shared property of cholinesterase inhibition. We used microarrays to characterize similarities and differences in transcriptional responses to chlorpyrifos and diazinon, assessing defined gene groupings for the pathways known to be associated with the mechanisms and/or outcomes of chlorpyrifos-induced developmental neurotoxicity. We exposed neonatal rats to daily doses of chlorpyrifos (1 mg/kg) or diazinon (1 or 2 mg/kg) on postnatal days 1–4 and evaluated gene expression profiles in brainstem and forebrain on day 5; these doses produce little or no cholinesterase inhibition. We evaluated pathways for general neural cell development, cell signaling, cytotoxicity and neurotransmitter systems, and identified significant differences for >60% of 252 genes. Chlorpyrifos elicited major transcriptional changes in genes involved in neural cell growth, development of glia and myelin, transcriptional factors involved in neural cell differentiation, cAMP-related cell signaling, apoptosis, oxidative stress, excitotoxicity, and development of neurotransmitter synthesis, storage and receptors for acetylcholine, serotonin, norepinephrine and dopamine. Diazinon had similar effects on many of the same processes but also showed major differences from chlorpyrifos. Our results buttress the idea that different organophosphates target multiple pathways involved in neural cell development but also that they deviate in key aspects that may contribute to disparate neurodevelopmental outcomes. Equally important, these pathways are compromised at exposures that are unrelated to biologically significant cholinesterase inhibition and its associated signs of systemic toxicity. The approach used here demonstrates how planned comparisons with microarrays can be used to screen for developmental neurotoxicity.

Glucocorticoid action networks and complex psychiatric and/or somatic disorders

Glucocorticoids contribute fundamentally to the maintenance of basal and stress-related homeostasis in all higher organisms. These hormones influence a large percentage of the expressed human genome and their effects spare almost no organs or tissues. Glucocorticoids influence many functions of the central nervous system, such as arousal, cognition, mood and sleep, the activity and direction of intermediary metabolism, the maintenance of a normal cardiovascular tone, the activity and quality of the immune and inflammatory reaction, including the manifestations of the sickness syndrome, as well as growth and reproduction. The numerous actions of glucocorticoids are mediated by a set of at least 16 glucocorticoid receptor (GR) isoforms forming homo- or hetero-dimers. The GRs consist of multifunctional domain proteins operating as ligand-dependent transcription factors that interact with many other cell signaling systems. The presence of multiple GR monomers and dimers expressed in a cell-specific fashion at different quantities with quantitatively and qualitatively different transcriptional activities suggests that the glucocorticoid signaling system is highly stochastic. Based on ample evidence, we present our conception that glucocorticoids are heavily involved in human pathophysiology and influence life expectancy. Common psychiatric and/or somatic complex disorders, such as anxiety, depression, insomnia, chronic pain and fatigue syndromes, obesity, the metabolic syndrome, essential hypertension, diabetes type 2, atherosclerosis with its cardiovascular sequelae, and osteoporosis, as well as autoimmune inflammatory and allergic disorders, all appear to have a glucocorticoid component.

Role of transcription factor AP-1 in integration of cell signaling systems

Role of transcription factor AP-1 in integration of cell signaling systems

A Mind for Structure: Exploring the Roots of Intelligent Systems

This book is an attempt to offer foundations for understanding the evolution and nature of cognitive functions. It argues that what has been missing is a full appreciation of the complexity of environmental change; how it furnishes important dynamic structure; and how the increasing complexity, and associated abstraction, of that structure, has driven the evolution of complex systems, especially of cognitive functions. The book shows how recent dynamic systems theory is leading to a better appreciation of environmental structure and how the origins of life itself are based on such structure. The book then goes on to illustrate evolving capacities for the abstraction of structure in genetic and epigenetic systems; in cell signalling and developmental systems; and in perceptual and cognitive systems, reaching a new potency in human socio-cognitive abilities. The idea has many implications for research and theory on the human mind; for the goals and purposes of science itself in such dynamic fields; and for the nature of interventions in many practical domains.

Extracellular heat shock protein 70 mediates heat stress-induced epidermal growth factor receptor transactivation in A431 carcinoma cells

The initial steps of heat stress in A431 cells were previously characterized by ligand-independent EGFR transactivation via an unknown mechanism and concomitant secretion of Hsp70. In this work we demonstrate that the depletion of Hsp70 from the conditioned medium of heated cells abolishes EGFR transactivation indicating that secreted Hsp70 is essential for EGFR transactivation during heat shock. This notion is supported by the findings that purified Hsp70 can induce EGFR transactivation and the activation of EGFR-dependent signaling pathways. Both heat stress and pure Hsp70 stimulate activation of TLR2/4 and their association with EGFR. These results suggest that the secreted Hsp70 mediates the cross-communication of TLR and EGFR signaling systems in A431 cells.

Molecular basis for control of conjugation by bacterial pheromone and inhibitor peptides

In many bacteria expression of lateral gene transfer and of virulence factors is controlled by cell-cell signalling systems. Molecular interactions of microbial signal molecules with their cognate receptors are not well understood. For the Enterococcus faecalis conjugative plasmid pCF10, the PrgX protein serves as a molecular switch controlling expression of conjugation and virulence genes encoded by the plasmid. The induction state of a pCF10-carrying donor cell is determined by the ratio of two signalling peptides, cCF10 pheromone and iCF10 inhibitor. Recent analysis of PrgX/cCF10 interactions suggests a mechanism for conversion to the induced state. However, the means by which iCF10 peptide antagonizes cCF10 activity is unclear, and it has been suggested that inhibitor peptides block import of pheromone peptides. We now show that both of these peptides interact with the same binding pocket of PrgX, but they differentially alter the conformation of the protein and its oligomerization state, resulting in opposing biological activities.

Porphyromonas gingivalisFimbriae Proactively Modulate β2Integrin Adhesive Activity and Promote Binding to and Internalization by Macrophages

In monocytes, the fimbriae of the oral pathogen Porphyromonas gingivalis activate cross talk signaling from Toll-like receptor 2 (TLR2) to the beta2 integrin CD11b/CD18, leading to the induction of the high-affinity state of the latter receptor. CD14 plays an important role in this "inside-out" proadhesive pathway by binding fimbriae and facilitating the activation of TLR2 and phosphatidylinositol 3-kinase signaling. In its high-affinity state, CD11b/CD18 mediates monocyte adhesion to endothelial cells and transmigration to sites of infection. We have now shown that P. gingivalis fimbriae function as both an activator and a ligand of CD11b/CD18; thus, fimbriae proactively promote their own binding to monocytes. Indeed, treatments that interfered with fimbria-induced activation of CD11b/CD18 (i.e., blockade of CD14, TLR2, or phosphatidylinositol 3-kinase signaling) also suppressed the cell binding activity of fimbriae, which was largely inducible and CD11b/CD18 dependent. Development of a recombinant inside-out signaling system in Chinese hamster ovary cells confirmed the ability of fimbriae to activate CD14/TLR2 signaling and induce their own CD11b/CD18-dependent binding. Induction of this proadhesive pathway by P. gingivalis fimbriae appeared to take place in lipid rafts. Indeed, methyl-beta-cyclodextrin, a cholesterol-sequestering agent that disrupts lipid raft organization, was found to inhibit the fimbria-induced assembly of CD14/TLR2 signaling complexes and the activation of the high-affinity state of CD11b/CD18. Experiments using macrophages from mice deficient in various pattern recognition receptors indicated that the receptors involved in the inside-out proadhesive pathway (CD14, TLR2, and CD11b/CD18) are important for mediating P. gingivalis internalization within macrophages. It therefore appears that P. gingivalis proactively modulates beta2 integrin adhesive activity for intracellular uptake.

Cytokine Receptors and Jak-STAT Signaling

This Teaching Resource provides lecture notes and slides for a class covering interferons, their receptors, and Jak-STAT signaling and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the different families of cytokines and their receptors and then proceeds to describe how cytokine receptor interactions lead to the activation of different members of the STAT family of transcription factors.

Space, Selection, and Surveillance: Setting Boundaries with BLyS

The BLyS family of ligands and receptors governs B cell homeostasis by controlling survival, differentiation, and lifespan. This family consists of multiple receptors and ligands, allowing independent regulation of different B cell subsets by varying the combination and levels of receptors expressed. Multiple downstream signaling pathways are implicated in these activities, reflecting this receptor complexity as well as cross-talk with other B cell signaling systems. BLyS levels are associated with multiple forms of humoral autoimmunity and can modulate tolerogenic elimination at the transitional checkpoint. BLyS responsiveness thus balances peripheral selection against cell numbers, providing an elastic system that varies selective stringency based on homeostatic demands.

Assembly and Organization of Macromolecular Complexes

This Teaching Resource provides lecture notes and slides for a class covering the roles of scaffold proteins in signal transduction and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture includes an overview of scaffold proteins in signal integration and focuses on the following scaffold proteins: A-kinase anchoring proteins (AKAPs), WAVE-1, and proteins that contain PDZ domains.

Identifying Illness Parameters in Fatiguing Syndromes Using Classical Projection Methods

To examine the potential of multivariate projection methods in identifying common patterns of change in clinical and gene expression data that capture the illness state of subjects with unexplained fatigue and nonfatigued control participants. Data for 111 female subjects was examined. A total of 59 indicators, including multidimensional fatigue inventory (MFI), medical outcome Short Form 36 (SF-36), Centers for Disease Control and Prevention (CDC) symptom inventory and cognitive response described illness. Partial least squares (PLS) was used to construct two feature spaces: one describing the symptom space from gene expression in peripheral blood mononuclear cells (PBMC) and one based on 117 clinical variables. Multiplicative scatter correction followed by quantile normalization was applied for trend removal and range adjustment of microarray data. Microarray quality was assessed using mean Pearson correlation between samples. Benjamini-Hochberg multiple testing criteria served to identify significantly expressed probes. A single common trend in 59 symptom constructs isolates of nonfatigued subjects from the overall group. This segregation is supported by two co-regulation patterns representing 10% of the overall microarray variation. Of the 39 principal contributors, the 17 probes annotated related to basic cellular processes involved in cell signaling, ion transport and immune system function. The single most influential gene was sestrin 1 (SESN1), supporting recent evidence of oxidative stress involvement in chronic fatigue syndrome (CFS). Dominant variables in the clinical feature space described heart rate variability (HRV) during sleep. Potassium and free thyroxine (T4) also figure prominently. Combining multiple symptom, gene or clinical variables into composite features provides better discrimination of the illness state than even the most influential variable used alone. Although the exact mechanism is unclear, results suggest a common link between oxidative stress, immune system dysfunction and potassium imbalance in CFS patients leading to impaired sympatho-vagal balance strongly reflected in abnormal HRV.

Regulation of Protein Translation

This Teaching Resource provides a summary and slides derived from a lecture on protein translation and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the various components that perform the translation process and then proceeds to describe the initiation, scanning, and ribosomal entry processes. The lecture concludes with the signaling mechanisms underlying translation regulation.