Signaling Intermediates Research Articles

Metabolic regulation of mitochondrial reactive oxygen species (mtROS) production and transient receptor potential vanilloid‐4 (TRPV4) activation in microvascular endothelial cells in PAH

Rationale In pulmonary arterial hypertension (PAH), highly proliferative microvascular endothelial cells (MVECs) are thought to contribute to vaso-occlusive lesion formation and increased pulmonary artery pressures. Using MVECs isolated from the Sugen/Hypoxia (SuHx) model of PAH, we recently showed that increased mtROS production activates the TRPV4 calcium channel and contributes to increased MVEC proliferation in vitro. However, the mechanistic basis for sustained mtROS production in MVECs isolated from SuHx rats (SuHx-MVECs) was unclear. Along with increased mtROS production, we observed evidence of significant mitochondrial dysfunction, including increased fragmentation and glycolytic shift. Since mtROS production can be increased by changes in the type of fuel (e.g. fatty acids)used to generate TCA metabolites, we hypothesized that use of non-glucose sources (specifically, increased fatty acid oxidation - FAO) could be the source of mitochondrial dysfunction and mtROS production in SuHx-MVECs. Methods Intracellular Ca2+ concentration ([Ca2+]i) was measured using Fura-2AM loaded MVEC in a flow chamber perfused with modified Krebs solution. Mitochondrial morphology was measured computationally using validated algorithms applied to confocal images of mitochondria in N- and SuHx-MVEC stained with MitoTracker. Lipid droplets were measured using BODIPY staining. Targeted metabolomics were performed on MVEC cell lysates using a standardized LC/MS approach as described previously (Suresh et al., AJP-Lung 2019). Human PAH metabolomics and genomics data were obtained from NIH Metabolomics Workbench or GEO Omnibus, respectively, and analyzed using R. Results Despite evidence of glycolytic shift (decreased oxygen consumption rate, increased extracellular acidification rate, increased extracellular lactate levels), levels of TCA metabolites, measured using targeted metabolomics, were not significantly lower in SuHx-MVECs compared to normoxic (N-MVEC) controls, suggesting presence of anaplerosis. Serum free fatty acids (FFA) were increased in SuHx rats at 5 weeks, while intracellular lipid droplet number was increased in SuHx-MVECs. Analysis of human PAH metabolomics and genomics data revealed increased serum FFA levels and increased expression of enzymes involved in breaking down long chain fatty acids for use in the TCA cycle (i.e. FAO). In SuHx-MVECs, untargeted metabolomics and metabolite-specific assays revealed increased levels of beta-hydroxybutyrate (BOHB), a ketone body produced by FAO. In N-MVECs, BOHB supplementation was sufficient to increase TRPV4 sensitivity to pharmacologic agonists. In contrast, inhibition of FAO (with Etoximir) in SuHx-MVECs attenuated basal Ca2+ and mitochondrial fragmentation to a similar extent as TRPV4 inhibition or mtROS quenching. Conclusion We conclude that intermediary signaling molecules such as BOHB and mtROS, produced as a consequence of increased FAO, act in concert to sensitize and activate the TRPV4 channel in lung MVECs. These data suggest an exciting role for metabolism as a regulator of TRPV4 channel activity in PAH.

TAK1 Inhibition Elicits Mitochondrial ROS to Block Intracellular Bacterial Colonization

Mitogen activated protein kinase kinase kinase 7 (MAP3K7), known as TAK1, is an intracellular signaling intermediate of inflammatory responses. However, a series of mouse Tak1 gene deletion analysis has revealed that ablation of TAK1 does not prevent but rather elicits inflammation, which is accompanied by elevation of reactive oxygen species (ROS). These have been considered as consequences of non-physiological loss of TAK1. Contrary to this view, here we propose that TAK1 inhibition-induced ROS are an active cellular process that targets intracellular bacteria. Intracellular bacterial effector proteins such as Yersinia's outer membrane protein YopJ are known to inhibit TAK1 to circumvent the inflammatory host responses. We found that such TAK1 inhibition particularly induces mitochondrial-derived ROS, which effectively destroys intracellular bacteria. Our results reveal a previously unrecognized host defense mechanism linking inhibition of inflammatory signaling with mitochondrial ROS.

A Growth Inhibitory PKCα‐ERK Signaling Pathway In the Intestine/Colon

Background Increasing evidence points to growth inhibitory and tumor suppressive roles of theprotein kinase C (PKC) family of serine/threonine kinases. The conventional PKC isozyme, PKCα, has anti-proliferative and antitumor effects in the epithelium of the intestine and colon. Activation of PKCα in intestinal cells induces a program of cell cycle withdrawal that involves downregulation of the pro-proliferative proteins cyclin D1 and Id1 and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. These growth inhibitory effects of PKCα are dependent on activation of the MEK-ERK pathway, a signaling axis with established pro-proliferative effects in normal intestinal cells and in colon cancer. The objective of this study is to define the novel PKCα-induced growth suppressive MEK-ERK signaling cascade identified in intestinal epithelial cells. Hypothesis We hypothesize that PKCα modulates the activity of unique signaling intermediates to activate growth inhibitory ERK signaling. We will establish the point at which PKCα intersects the RAS-RAF-MEK-ERK pathway to induce growth arrest, identify the factors that link PKCα to the ERK pathway, and define effects that determine antiproliferative vs proliferative ERK signaling in intestinal cells. Results We have determined that PKCα activates RAS, RAF, MEK, and ERK in intestinal epithelial cells. The requirement for each of these pathway components in PKCα-mediated modulation of cyclin D1, Id1, and p21Cip1 and cell cycle withdrawal was confirmed using genetic approaches and inhibition of RAS, RAF, MEK, and ERK activity with the pharmacological agents Salirasib, LY3009120, PD0325901, and SCH772984, respectively. PKCα activation was shown to induce dimerization/activation of all three members of the RAF family (ARAF, BRAF and CRAF) and combinatorial knockdown indicated redundancy between these proteins. siRNA-mediated knockdown excluded a role for the RAF-MEK-ERK scaffolding protein KSR1. Analysis of the roles of Ras guanine nucleotide exchange factors identified at least two growth inhibitory pathways downstream of PKCα. Knockdown of the RasGRP isoform, RasGRP3, but not RasGRP1 or 2, inhibited the ability of PKCα activation to upregulate p21Cip1. However, RasGRP3 knockdown had no effect on PKCα-induced downregulation of cyclin D1 or Id1. Consistent with these findings, knockdown of RasGRP3 only partially inhibited PKCα-induced growth arrest. Based on evidence that SOS1 phosphorylation status is altered by PKCα signaling, we are currently exploring the potential involvement of SOS1 in the growth-inhibitory effects of PKCα. Conclusion PKCα activates RAS upstream of RAF, MEK and ERK to induce cell cycle arrest in intestinal cells. Activation of growth inhibitory ERK signaling by PKCα involves at least two mechanisms, one of which is RasGRP3-dependent and leads to p21Cip1 induction. Ongoing studies using siRNA knockdown strategies are exploring the role of Ras-GEFs such as RasGRP3, SOS1 and SOS2 in mediating PKCα-induced activation of RAS, to identify distinctive effects related to growth inhibitory versus growth promoting ERK signaling.

Regulation of Epidermal Growth Factor Receptor by Clathrin‐Associated Kinases

The Epidermal Growth Factor (EGF) Receptor (EGFR) is a receptor tyrosine kinase (RTK) that controls many key components of cell physiology, including proliferation, survival, and metabolism. When upregulated, EGFR drives tumor growth and progression in several types of cancer. EGF stimulation elicits EGFR phosphorylation and activation of phosphatidylinositol-3-kinase (PI3K), leading to Akt activation by its phosphorylation on T308 and S473. EGF-stimulated Akt phosphorylation is dependent on clathrin coated pits (CCPs) at the plasma membrane, but not receptor endocytosis1. CCPs are 50-100 nm protein assemblies that are well-known endocytic portals and lead to eventual receptor downregulation. Importantly, we previously uncovered that CCPs are also required for Akt activation, likely by acting as protein and lipid scaffolds to coordinate signaling intermediates. One of the many proteins that are known to bind to clathrin directly is Activated Cdc42 Kinase 1 (Ack1), a non-receptor tyrosine kinase implicated in oncogenic RTK signaling and tumor cell survival. Ack1 also interacts with EGFR and directly phosphorylates Akt at Y176, a regulatory site distinct from those required for canonical Akt activation. In addition to clathrin, Ack1 also interacts with a multitude of other proteins. How Ack1 regulates the PI3K/Akt signaling pathway, and how binding of Ack1 to clathrin controls this phenomenon, remains poorly defined. Here, we examine how Ack1 contributes to EGFR signaling. Using siRNA silencing of Ack1 and/or the related isoform Ack2, alone or in combination with inducible expression of fluorescent or mutant Ack constructs, we examined the role of Ack1 in EGFR signaling. Moreover, using various microscopy and image analysis techniques, we examined the mechanism of recruitment of Ack1 to CCPs. Our results indicate a critical role for Ack1 in EGFR signaling, in particular for the activation of PI3K-Akt signaling. The improved understanding of the regulation and outcome of EGFR signals by proteins such as Ack1 and scaffolds such as clathrin-coated pits, can lead to the development of novel cancer treatments.

TRAF3 Interacting Protein 2 (TRAF3IP2) Expression is Increased in Failing Human Hearts of Ischemic Origin and Mediates the Pro‐fibrotic Responses of Transforming Growth Factor‐β (TGF‐β) in Adult Human Cardiac Fibroblasts

Heart failure (HF) is a leading cause of morbidity and mortality worldwide. Chronic inflammation contributes to HF development and progression. We have previously demonstrated the causal role and the therapeutic potential of targeting TRAF3IP2, a proinflammatory cytoplasmic adapter molecule, in ischemia/reperfusion-induced adverse myocardial remodeling and heart failure development in a mouse model. However, it is not known whether failing human hearts of ischemic origin express increased TRAF3IP2. Therefore, we hypothesized that TRAF3IP2 expression is increased during human HF of ischemic origin and investigated the molecular mechanisms underlying the induction and role of TRAF3IP2 in TGF-β-induced pro-fibrotic responses in cultured primary human cardiac fibroblasts (CF). The expression of TRAF3IP2 and its downstream signaling intermediates were analyzed in left ventricular (LV) tissue from explanted human hearts (failing human hearts of ischemic origin, HF-I) of both sexes, with LV tissue from non-failing (NF) human donor hearts deemed unsuitable for transplantation serving as controls. The data show that HF-I is characterized by myocardial hypertrophy, fibrosis (Picrosirius Red staining), contractile dysfunction (echocardiography), increased TRAF3IP2 expression (Western blotting and immunofluorescence), activation of Nuclear Factor Kappa B (NF-κB, phospho-p65), Activator Protein 1 (AP-1, phospho-c-Jun), and p38 Mitogen-Activated Protein Kinase (p38 MAPK, phospho-p38) (Western blotting using activation-specific antibodies), and enhanced expression of pro-inflammatory and pro-fibrotic mediators Interleukin-6 (IL-6), IL-17A, IL-18, Connective Tissue Growth Factor (CTGF) (RT-qPCR), collagens I and III, lysyl oxidase, Matrix Metalloproteinases (MMPs) (Western blotting), and TGF-β (functional assay). In cultured CF, TGF-β induced TRAF3IP2 expression in part via AP-1, and induced TRAF3IP2-dependent inflammatory cytokine expression, MMP2/9 activation, collagen expression, and migration (Boyden chamber assay) and proliferation (CyQuant assay). Moreover, forced expression of TRAF3IP2 by itself induced TGF-β expression. These data suggest that TRAF3IP2 plays a causal role in the development and progression of heart failure, and may be a therapeutic target.

KLF11 deficiency exacerbates renal damage in experimental unilateral ureteral obstruction

KLFs (Kruppel-like factors) comprise a family of over 17 distinct transcription factors that have been shown to regulate cell metabolism and renal function, including podocyte differentiation and maintenance of the glomerular filtration barrier. In non-renal systems, KLF11 has been shown to regulate fibrosis. However, no previous studies have established a potential role for KLF11 in renal pathophysiology. We sought to test the hypothesis that KLF11 regulates renal fibrosis in unilateral ureteral obstruction, a well-established murine model of renal fibrosis. Mice with homozygous deletion of the KLF11 gene (KO) and wild-type (WT) mice were subjected to unilateral ureteral obstruction (UUO; n=8 per group); sham controls (n=4 per group) were subjected to manipulation of the ureter without ligation. Kidneys were harvested at 9 days following surgery,. Semiquantitative histopathologic analysis of interstitial fibrosis, tubular atrophy, and interstitial inflammation was performed in a blinded fashion. Analysis of gene expression was performed on renal cortex using Pathway Detect RNA arrays and by RNASeq. Histopathologic analysis of sham controls revealed no significant alterations. However, expression of several elements of the TGF-beta signaling pathway was significantly higher in KLF11 KO sham mice, compared to WT mice, including TGF-beta receptors type I and type II, Smad2, Smad 7, Bmpr1B, and Bmpr2R, and the KLF-related transcription factor Sp1 (all p<0.05). In mice subjected to UUO, interstitial fibrosis, tubular atrophy, and interstitial inflammation were significantly greater in KLF11 KO mice compared to WT mice. Interstitial inflammation was associated with influx of alternatively activated macrophages, as assessed by quantitative analysis of CD206+ infiltrating cells (p<0.05); no significant increase in CD3+ T cell numbers were noted. Expression of several members of the TGF-beta signaling pathway were significantly increased in KLF11 KO UUO mice, compared to WT UUO mice, including TGF-beta 1, TGF-beta-2, and TGF-beta3, TGF-beta receptors type I and type II, and Smad 1,2,3,4, and 5 (all p<0.05). Conversely, expression of the transcription factor JunB was markedly increased in WT UUO mice compared to WT sham mice (12.2 -fold; p<0.0001). However, JunB expression was markedly decreased in KLF11 KO mice (both sham and UUO). Although MMP expression was not significantly different in KO versus WT mice, TIMP3 expression was higher in KO mice compared to WT mice. In conclusion, we found that renal damage is exacerbated in KLF11 KO mice subjected to UUO, compared to WT UUO mice and is associated with influx of potentially pro-fibrotic, alternatively activated M2 macrophages. KLF11 appears to be a critical regulator of TGF-beta signaling, as KLF11 deficiency is associated with increased expression of TGF-beta and signaling intermediates of this fibrogenic pathway.

Intrinsically Connected: Therapeutically Targeting the Cathepsin Proteases and the Bcl-2 Family of Protein Substrates as Co-regulators of Apoptosis.

Taken with the growing importance of cathepsin-mediated substrate proteolysis in tumor biology and progression, the focus and emphasis placed on therapeutic design and development is coming into fruition. Underpinning this approach is the invariable progression from the direction of fully characterizing cathepsin protease members and their substrate targets, towards targeting such an interaction with tangible therapeutics. The two groups of such substrates that have gained much attention over the years are the pro- and anti- apoptotic protein intermediates from the extrinsic and intrinsic signaling arms of the apoptosis pathway. As proteins that are central to determining cellular fate, some of them present themselves as very favorable candidates for therapeutic targeting. However, considering that both anti- and pro- apoptotic signaling intermediates have been reported to be downstream substrates for certain activated cathepsin proteases, therapeutic targeting approaches based on greater selectivity do need to be given greater consideration. Herein, we review the relationships shared by the cathepsin proteases and the Bcl-2 homology domain proteins, in the context of how the topical approach of adopting ‘BH3-mimetics’ can be explored further in modulating the relationship between the anti- and pro- apoptotic signaling intermediates from the intrinsic apoptosis pathway and their upstream cathepsin protease regulators. Based on this, we highlight important future considerations for improved therapeutic design.

Pepper NAC-type transcription factor NAC2c balances the trade-off between growth and defense responses.

Plant responses to pathogen attacks and high-temperature stress (HTS) are distinct in nature but generally share several signaling components. How plants produce specific responses through these common signaling intermediates remains elusive. With the help of reverse-genetics approaches, we describe here the mechanism underlying trade-offs in pepper (Capsicum annuum) between growth, immunity, and thermotolerance. The NAC-type transcription factor CaNAC2c was induced by HTS and Ralstonia solanacearum infection (RSI). CaNAC2c-inhibited pepper growth, promoted immunity against RSI by activating jasmonate-mediated immunity and H2O2 accumulation, and promoted HTS responses by activating Heat shock factor A5 (CaHSFA5) transcription and blocking H2O2 accumulation. We show that CaNAC2c physically interacts with CaHSP70 and CaNAC029 in a context-specific manner. Upon HTS, CaNAC2c-CaHSP70 interaction in the nucleus protected CaNAC2c from degradation and resulted in the activation of thermotolerance by increasing CaNAC2c binding and transcriptional activation of its target promoters. CaNAC2c did not induce immunity-related genes under HTS, likely due to the degradation of CaNAC029 by the 26S proteasome. Upon RSI, CaNAC2c interacted with CaNAC029 in the nucleus and activated jasmonate-mediated immunity but was prevented from activating thermotolerance-related genes. In non-stressed plants, CaNAC2c was tethered outside the nucleus by interaction with CaHSP70, and thus was unable to activate either immunity or thermotolerance. Our results indicate that pepper growth, immunity, and thermotolerance are coordinately and tightly regulated by CaNAC2c via its inducible expression and differential interaction with CaHSP70 and CaNAC029.

Sex differences in synaptic plasticity underlying learning.

Although sex differences in learning behaviors are well documented, sexual dimorphism in the synaptic processes of encoding is only recently appreciated. Studies in male rodents have built upon the discovery of long-term potentiation (LTP), and acceptance of this activity-dependent increase in synaptic strength as a mechanism of encoding, to identify synaptic receptors and signaling activities that coordinate the activity-dependent remodeling of the subsynaptic actin cytoskeleton that is critical for enduring potentiation and memory. These molecular substrates together with other features of LTP, as characterized in males, have provided an explanation for a range of memory phenomena including multiple stages of consolidation, the efficacy of spaced training, and the location of engrams at the level of individual synapses. In the present report, we summarize these findings and describe more recent results from our laboratories showing that in females the same actin regulatory mechanisms are required for hippocampal LTP and memory but, in females only, the engagement of both modulatory receptors such as TrkB and synaptic signaling intermediaries including Src and ERK1/2 requires neuron-derived estrogen and signaling through membrane-associated estrogen receptor α (ERα). Moreover, in association with the additional ERα involvement, females exhibit a higher threshold for hippocampal LTP and spatial learning. We propose that the distinct LTP threshold in females contributes to as yet unappreciated sex differences in information processing and features of learning and memory.

Recent advances and health implications of dietary fasting regimens on the gut microbiome.

Calorie restriction is a primary dietary intervention demonstrated over many decades in cellular and animal models to modulate aging pathways, positively affect age-associated diseases and, in clinical studies, to promote beneficial health outcomes. Because long-term compliance with daily calorie restriction has proven problematic in humans several intermittent fasting regimens, including alternate day fasting and time-restricted feeding, have evolved revealing similar clinical benefits as calorie restriction. Despite significant research on the cellular and physiological mechanisms contributing to, and responsible for, these observed benefits, relatively little research has investigated the impact of these various fasting protocols on the gut microbiome (GM). Reduced external nutrient supply to the gut may beneficially alter the composition and function of a "fed" gut microflora. Indeed, the prevalent, obesogenic Western diet can promote deleterious changes in the GM, signaling intermediates involved in lipid and glucose metabolism, and immune responses in the gastrointestinal tract. This review describes recent preclinical and clinical effects of varying fasting regimens on GM composition and associated physiology. Although the number of preclinical and clinical interventions are limited, significant data thus far suggest fasting interventions impact GM composition and physiology. However, there are considerable heterogeneities of study design, methodological considerations, and practical implications. Ongoing research on the health impact of fasting regimens on GM modulation is warranted.

CD105 (Endoglin): A Potential Anticancer Therapeutic Inhibits Mitogenesis and Map Kinase Pathway Activation.

CD105 is highly expressed on human activated endothelial cells (ECs), is an important component of the TGF-β1 receptor complex and is essential for angiogenesis. CD105 expression is up-regulated in activated ECs and is an important potential marker for cancer prognosis. In vitro rat myoblasts transfected with the L-CD105 and S-CD105 transfectants. The transfectants were treated with TGF-β1 for the angiogenesis study. L-CD105 affects cell proliferation in the presence and absence of TGF-β1, and inhibits p-ERK1/2, p-MEK1/2 and p-c-Jun in L-CD105 transfectants compared to controls. The induction of phospho-ERK1/2 following treatment with TGF-β1 remained significantly lower in L-CD105 transfectants compared to controls. L-CD105 inhibits the phosphorylation of ERK1/2, MEK1/2, c-Jun1/2/3, and associated signalling intermediates. CD105 modulates cell growth and TGF-β1 induced cell signalling through ERK-c-Jun expression.

Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-β which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.

Spatiotemporal Dynamics of RAC1 Signaling During Wound Healing in Forster Resonance Energy Transfer Biosensor Mice

The advent of Förster Resonance Energy Transfer (FRET) biosensor transgenic animals presents the unique opportunity to spatially specify and quantify the level of active signal transduction within individual cells of a tissue with subcellular resolution. The type of information gleaned by these biosensors are key for understanding the dynamic role of signaling intermediates in regulating cellular plasticity during developmental and regenerative processes. The modified Raichu-Rac1 biosensors transgenically encoded in mice FRET when bound to GTP and report active signal transduction.

Synthetic VSMCs Induce BBB Disruption Mediated by MYPT1 in Ischemic Stroke

Vascular smooth muscle cells (VSMCs) have been widely recognized as key players in regulating blood-brain barrier (BBB) function, their roles are unclear in ischemic stroke. Myosin phosphatase target subunit 1 (MYPT1) is essential for VSMCs contraction and contributes to maintaining healthy vasculature. We generated VSMC-specific MYPT1 knockout mice (MYPT1SMKO mice) and cultured VSMCs infected with Lv-shMYPT1 to explore BBB integrity, brain infarction and neurological deficits after ischemic stroke. MYPT1 deficiency induced phenotypic switching of synthetic VSMCs, which aggravated BBB disruption. Proteomic analysis identified evolutionarily conserved signaling intermediates in Toll pathways (ECSIT) as a downstream molecule that promotes phenotypic switching of VSMCs and contributed to IL-6 expression. Knocking down ECSIT rescued the phenotypic switching of VSMCs and BBB disruption in ischemic stroke. Additionally, inhibition of IL-6 decreased BBB permeability. These findings reveal that MYPT1 deficiency activated phenotypic switching of VSMCs and induced BBB disruption after ischemic stroke through ECSIT-IL-6 signaling.

A DYRK1B-dependent pathway suppresses rDNA transcription in response to DNA damage.

DNA double-strand breaks (DSBs) at ribosomal gene loci trigger inhibition of ribosomal DNA (rDNA) transcription and extensive nucleolar reorganization, including the formation of nucleolar caps where rDNA DSBs engage with canonical DSB signaling and repair factors. While these nucleolar responses underlie maintenance of rDNA stability, the molecular components that drive each of these events remain to be defined. Here we report that full suppression of rRNA synthesis requires the DYRK1B kinase, a nucleolar DSB response that can be uncoupled from ATM-mediated DSB signaling events at the nucleolar periphery. Indeed, by targeting DSBs onto rDNA arrays, we uncovered that chemical inhibition or genetic inactivation of DYRK1B led to sustained nucleolar transcription. Not only does DYRK1B exhibit robust nucleolar accumulation following laser micro-irradiation across cell nuclei, we further showed that DYRK1B is required for rDNA DSB repair and rDNA copy number maintenance, and that DYRK1B-inactivated cells are hypersensitised to DSBs induced at the rDNA arrays. Together, our findings not only identify DYRK1B as a key signaling intermediate that coordinates DSB repair and rDNA transcriptional activities, but also support the idea of specialised DSB responses that operate within the nucleolus to preserve rDNA integrity.

Devil in the details: Mechanistic variations impact information transfer across models of transcriptional cascades.

The transcriptional network determines a cell’s internal state by regulating protein expression in response to changes in the local environment. Due to the interconnected nature of this network, information encoded in the abundance of various proteins will often propagate across chains of noisy intermediate signaling events. The data-processing inequality (DPI) leads us to expect that this intracellular game of “telephone” should degrade this type of signal, with longer chains losing successively more information to noise. However, a previous modeling effort predicted that because the steps of these signaling cascades do not truly represent independent stages of data processing, the limits of the DPI could seemingly be surpassed, and the amount of transmitted information could actually increase with chain length. What that work did not examine was whether this regime of growing information transmission was attainable by a signaling system constrained by the mechanistic details of more complex protein-binding kinetics. Here we address this knowledge gap through the lens of information theory by examining a model that explicitly accounts for the binding of each transcription factor to DNA. We analyze this model by comparing stochastic simulations of the fully nonlinear kinetics to simulations constrained by the linear response approximations that displayed a regime of growing information. Our simulations show that even when molecular binding is considered, there remains a regime wherein the transmitted information can grow with cascade length, but ends after a critical number of links determined by the kinetic parameter values. This inflection point marks where correlations decay in response to an oversaturation of binding sites, screening informative transcription factor fluctuations from further propagation down the chain where they eventually become indistinguishable from the surrounding levels of noise.

Stimulation of Vibratory Urticaria-Associated Adhesion-GPCR, EMR2/ADGRE2, Triggers the NLRP3 Inflammasome Activation Signal in Human Monocytes.

EMR2/ADGRE2 is an adhesion G protein-coupled receptor differentially expressed by human myeloid cells. It modulates diverse cellular functions of innate immune cells and a missense EMR2 variant is directly responsible for vibratory urticaria. Recently, EMR2 was found to activate NLRP3 inflammasome in monocytes via interaction with FHR1, a regulatory protein of complement Factor H. However, the functional involvement of EMR2 activation and its signaling mechanisms in eliciting NLRP3 inflammasome activation remain elusive. In this study, we show that EMR2-mediated signaling plays a critical role in triggering the activation (2nd) signal for the NLRP3 inflammasome in both THP-1 monocytic cell line and primary monocytes. Stimulation of EMR2 by its agonistic 2A1 monoclonal antibody elicits a Gα16-dependent PLC-β activation pathway, inducing the activity of downstream Akt, MAPK, NF-κB, and Ca2+ mobilization, eventually leading to K+ efflux. These results identify EMR2 and its associated signaling intermediates as potential intervention targets of NLRP3 inflammasome activation in inflammatory disorders.

E3 Ubiquitin Ligases: Key Regulators of TGFβ Signaling in Cancer Progression.

Transforming growth factor β (TGFβ) is a secreted growth and differentiation factor that influences vital cellular processes like proliferation, adhesion, motility, and apoptosis. Regulation of the TGFβ signaling pathway is of key importance to maintain tissue homeostasis. Perturbation of this signaling pathway has been implicated in a plethora of diseases, including cancer. The effect of TGFβ is dependent on cellular context, and TGFβ can perform both anti- and pro-oncogenic roles. TGFβ acts by binding to specific cell surface TGFβ type I and type II transmembrane receptors that are endowed with serine/threonine kinase activity. Upon ligand-induced receptor phosphorylation, SMAD proteins and other intracellular effectors become activated and mediate biological responses. The levels, localization, and function of TGFβ signaling mediators, regulators, and effectors are highly dynamic and regulated by a myriad of post-translational modifications. One such crucial modification is ubiquitination. The ubiquitin modification is also a mechanism by which crosstalk with other signaling pathways is achieved. Crucial effector components of the ubiquitination cascade include the very diverse family of E3 ubiquitin ligases. This review summarizes the diverse roles of E3 ligases that act on TGFβ receptor and intracellular signaling components. E3 ligases regulate TGFβ signaling both positively and negatively by regulating degradation of receptors and various signaling intermediates. We also highlight the function of E3 ligases in connection with TGFβ’s dual role during tumorigenesis. We conclude with a perspective on the emerging possibility of defining E3 ligases as drug targets and how they may be used to selectively target TGFβ-induced pro-oncogenic responses.

Charting protein dephosphorylation triggered by Toll-like receptor (TLR) signaling in macrophages and its role in health and disease.

Toll-like receptor (TLR) signaling induces substantial changes in the phosphoproteome of innate immune cells, mainly in the form of increased phosphorylation of signaling intermediaries. Loss of constitutive phosphorylation occurs simultaneously, but these transitions from a stable, phosphorylated state in resting cells to a sustained underphosphorylated state in activated cells have received far less attention. This review provides an overview of phosphorylation sites downregulated during TLR-mediated signaling, with a particular focus on TLR4 activation by lipopolysaccharide (LPS). Energy homeostasis, the cell cycle, mitochondrial fission, and gene regulation are among the biological events in macrophages that are regulated through the downregulation of phosphoproteins as part of intracellular signaling events. Phosphoproteomics studies on innate immune cells have identified hundreds of hitherto uncharacterized phosphorylation sites that are lost upon stimulation, indicating that protein hypophosphorylation is a significant, largely unexplored layer of complexity in the TLR4 pathway.

Heat Shock Protein-90 Inhibition Alters Activation of Pancreatic Stellate Cells and Enhances the Efficacy of PD-1 Blockade in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a prominent fibrotic stroma, which is a result of interactions between tumor, immune and pancreatic stellate cells (PSC), or cancer-associated fibroblasts (CAF). Targeting inflammatory pathways present within the stroma may improve access of effector immune cells to PDAC and response to immunotherapy. Heat shock protein-90 (Hsp90) is a chaperone protein and a versatile target in pancreatic cancer. Hsp90 regulates a diverse array of cellular processes of relevance to both the tumor and the immune system. However, to date the role of Hsp90 in PSC/CAF has not been explored in detail. We hypothesized that Hsp90 inhibition would limit inflammatory signals, thereby reprogramming the PDAC tumor microenvironment to enhance sensitivity to PD-1 blockade. Treatment of immortalized and primary patient PSC/CAF with the Hsp90 inhibitor XL888 decreased IL6, a key cytokine that orchestrates immune changes in PDAC at the transcript and protein level in vitro XL888 directly limited PSC/CAF growth and reduced Jak/STAT and MAPK signaling intermediates and alpha-SMA expression as determined via immunoblot. Combined therapy with XL888 and anti-PD-1 was efficacious in C57BL/6 mice bearing syngeneic subcutaneous (Panc02) or orthotopic (KPC-Luc) tumors. Tumors from mice treated with both XL888 and anti-PD-1 had a significantly increased CD8+ and CD4+ T-cell infiltrate and a unique transcriptional profile characterized by upregulation of genes associated with immune response and chemotaxis. These data demonstrate that Hsp90 inhibition directly affects PSC/CAF in vitro and enhances the efficacy of anti-PD-1 blockade in vivo.