Genaro Vázquez-Victorio

Vesicular Trafficking and Signaling for Cytokine and Chemokine Secretion in Mast Cells

Upon activation mast cells (MCs) secrete numerous inflammatory compounds stored in their cytoplasmic secretory granules by a process called anaphylactic degranulation, which is responsible for type I hypersensitivity responses. Prestored mediators include histamine and MC proteases but also some cytokines and growth factors making them available within minutes for a maximal biological effect. Degranulation is followed by the de novo synthesis of lipid mediators such as prostaglandins and leukotrienes as well as a vast array of cytokines, chemokines, and growth factors, which are responsible for late phase inflammatory responses. While lipid mediators diffuse freely out of the cell through lipid bilayers, both anaphylactic degranulation and secretion of cytokines, chemokines, and growth factors depends on highly regulated vesicular trafficking steps that occur along the secretory pathway starting with the translocation of proteins to the endoplasmic reticulum. Vesicular trafficking in MCs also intersects with endocytic routes, notably to form specialized cytoplasmic granules called secretory lysosomes. Some of the mediators like histamine reach granules via specific vesicular monoamine transporters directly from the cytoplasm. In this review, we try to summarize the available data on granule biogenesis and signaling events that coordinate the complex steps that lead to the release of the inflammatory mediators from the various vesicular carriers in MCs.

RETRACTED: 6-OHDA-induced apoptosis and mitochondrial dysfunction are mediated by early modulation of intracellular signals and interaction of Nrf2 and NF-κB factors

6-Hydroxydopamine (6-OHDA) is a neurotoxin that generates an experimental model of Parkinsons disease in rodents and is commonly employed to induce a lesion in dopaminergic pathways. The characterization of those molecular mechanisms linked to 6-OHDA-induced early toxicity is needed to better understand the cellular events further leading to neurodegeneration. The present work explored how 6-OHDA triggers early downstream signaling pathways that activate neurotoxicity in the rat striatum. Mitochondrial function, caspases-dependent apoptosis, kinases signaling (Akt, ERK 1/2, SAP/JNK and p38) and crosstalk between nuclear factor kappa B (NF-κB) and nuclear factor-erythroid-2-related factor 2 (Nrf2) were evaluated at early times post-lesion. We found that 6-OHDA initiates cell damage via mitochondrial complex I inhibition, cytochrome c and apoptosis-inducing factor (AIF) release, as well as activation of caspases 9 and 3 to induce apoptosis, kinase signaling modulation and NF-κB-mediated inflammatory responses, accompanied by inhibition of antioxidant systems regulated by the Nrf2 pathway. Our results suggest that kinases SAP/JNK and p38 up-regulation may play a role in the early stages of 6-OHDA toxicity to trigger intrinsic pathways for apoptosis and enhanced NF-κB activation. In turn, these cellular events inhibit the activation of cytoprotective mechanisms, thereby leading to a condition of general damage.

S-allyl cysteine protects against 6-hydroxydopamine-induced neurotoxicity in the rat striatum: involvement of Nrf2 transcription factor activation and modulation of signaling kinase cascades.

Pharmacological activation at the basal ganglia of the transcription factor Nrf2, guardian of redox homeostasis, holds a strong promise for the slow progression of Parkinsons disease (PD). However, a potent Nrf2 activator in the brain still must be found. In this study, we have investigated the potential use of the antioxidant compound S-allyl cysteine (SAC) in the activation of Nrf2 in 6-hydoxydopamine (6-OHDA)-intoxicated rats. In the rat striatum, SAC by itself promoted the Nrf2 dissociation of Keap-1, its nuclear translocation, the subsequent association with small MafK protein, and further binding of the Nrf2/MafK complex to ARE sequence, as well as the up-regulation of Nrf2-dependent genes encoding the antioxidant enzymes HO-1, NQO-1, GR, and SOD-1. In vivo and in vitro experiments to identify signaling pathways activated by SAC pointed to Akt as the most likely kinase participating in Nrf2 activation by SAC. In PC12 cells, SAC stimulated the activation of Akt and ERK1/2 and inhibited JNK1/2/3 activation. In the rat striatum, the SAC-induced activation of Nrf2 is likely to contribute to inhibit the toxic effects of 6-OHDA evidenced by phase 2 antioxidant enzymes up-regulation, glutathione recovery, and attenuation of reactive oxygen species (ROS), nitric oxide (NO), and lipid peroxides formation. These early protective effects correlated with the long-term preservation of the cellular redox status, the striatal dopamine (DA) and tyrosine hydroxylase (TH) levels, and the improvement of motor skills. Therefore, this study indicates that, in addition to direct scavenging actions, the activation of Nrf2 by SAC might confer neuroprotective responses through the modulation of kinase signaling pathways in rodent models of PD, and suggests that this antioxidant molecule may have a therapeutic value in this human pathology.

Transforming growth factor-β/SMAD Target gene SKIL is negatively regulated by the transcriptional cofactor complex SNON-SMAD4.

Background: Human SKIL gene encodes for SNON, a negative regulator of the TGF-β/SMAD pathway. Results: We provide a molecular mechanism of transcriptional regulation of SKIL gene expression by TGF-β/SMADs. Conclusion: Transcriptional cofactor complex SNON-SMAD4 negatively controls the expression of SKIL gene. Significance: The formation and function of complex SNON-SMAD4 are impaired in cancer cells lacking SMAD4, which affects TGF β-target gene regulation. The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-β signaling. SNON protein levels are tightly regulated by the TGF-β pathway: whereas a short stimulation with TGF-β decreases SNON levels by its degradation via the proteasome, longer TGF-β treatment increases SNON levels by inducing SKIL gene expression. Here, we investigated the molecular mechanisms involved in the self-regulation of SKIL gene expression by SNON. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-β response element (TRE) bearing four groups of SMAD-binding elements that are also conserved in mouse. Two regions of 408 and 648 bp of the human SKIL gene (∼2.4 kb upstream of the ATG initiation codon) containing the core promoter, transcription start site, and the TRE were cloned for functional analysis. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-β treatment was demonstrated by ChIP and sequential ChIP assays. Interestingly, the SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. In response to TGF-β signal, SNON is removed from the SKIL gene promoter, and then the activated SMAD complexes bind the promoter to induce SKIL gene expression. Subsequently, the up-regulated SNON protein in complex with SMAD4 represses its own expression as part of the negative feedback loop regulating the TGF-β pathway. Accordingly, when the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-β target genes is modified.

Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes

Background: The Ski oncoprotein and tumor suppressor is a negative regulator of the antimitotic TGF-β/Smad pathway. Results: The Ski protein is localized in the nucleus and cytoplasm of hepatocytes. Ski protein stability is controlled differentially by actin cytoskeleton dynamics. Conclusion: TGF-β/Smads and GPCR/actin cytoskeleton-dynamic signals regulate Ski protein stability via the proteasome in hepatocytes. Significance: Stabilization of Ski protein may favor the proliferation of regenerating hepatocyte. TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

GPCRs and actin-cytoskeleton dynamics.

A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others. This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study.

Protein Tyrosine Kinase Fyn Regulates TLR4-Elicited Responses on Mast Cells Controlling the Function of a PP2A-PKCα/β Signaling Node Leading to TNF Secretion

Mast cells produce proinflammatory cytokines in response to TLR4 ligands, but the signaling pathways involved are not fully described. In this study, the participation of the Src family kinase Fyn in the production of TNF after stimulation with LPS was evaluated using bone marrow–derived mast cells from wild-type and Fyn-deficient mice. Fyn−/− cells showed higher LPS-induced secretion of preformed and de novo–synthesized TNF. In both cell types, TNF colocalized with vesicle-associated membrane protein (VAMP)3-positive compartments. Addition of LPS provoked coalescence of VAMP3 and its interaction with synaptosomal-associated protein 23; those events were increased in the absence of Fyn. Higher TNF mRNA levels were also observed in Fyn-deficient cells as a result of increased transcription and greater mRNA stability after LPS treatment. Fyn−/− cells also showed higher LPS-induced activation of TAK-1 and ERK1/2, whereas IκB kinase and IκB were phosphorylated, even in basal conditions. Increased responsiveness in Fyn−/− cells was associated with a lower activity of protein phosphatase 2A (PP2A) and augmented activity of protein kinase C (PKC)α/β, which was dissociated from PP2A and increased its association with the adapter protein neuroblast differentiation–associated protein (AHNAK, desmoyokin). LPS-induced PKCα/β activity was associated with VAMP3 coalescence in WT and Fyn-deficient cells. Reconstitution of MC-deficient Wsh mice with Fyn−/− MCs produced greater LPS-dependent production of TNF in the peritoneal cavity. Our data show that Fyn kinase is activated after TLR4 triggering and exerts an important negative control on LPS-dependent TNF production in MCs controlling the inactivation of PP2Ac and activation of PKCα/β necessary for the secretion of TNF by VAMP3+ carriers.

Actin-cytoskeleton polymerization differentially controls the stability of Ski and SnoN co-repressors in normal but not in transformed hepatocytes.

BACKGROUND Ski and SnoN proteins function as transcriptional co-repressors in the TGF-β pathway. They regulate cell proliferation and differentiation, and their aberrant expression results in altered TGF-β signalling, malignant transformation, and alterations in cell proliferation. METHODS We carried out a comparative characterization of the endogenous Ski and SnoN protein regulation by TGF-β, cell adhesion disruption and actin-cytoskeleton rearrangements between normal and transformed hepatocytes; we also analyzed Ski and SnoN protein stability, subcellular localization, and how their protein levels impact the TGF-β/Smad-driven gene transcription. RESULTS Ski and SnoN protein levels are lower in normal hepatocytes than in hepatoma cells. They exhibit a very short half-life and a nuclear/cytoplasmic distribution in normal hepatocytes opposed to a high stability and restricted nuclear localization in hepatoma cells. Interestingly, while normal cells exhibit a transient TGF-β-induced gene expression, the hepatoma cells are characterized by a strong and sustained TGF-β-induced gene expression. A novel finding is that Ski and SnoN stability is differentially regulated by cell adhesion and cytoskeleton rearrangements in the normal hepatocytes. The inhibition of protein turnover down-regulated both Ski and SnoN co-repressors impacting the kinetic of expression of TGF-β-target genes. CONCLUSION Normal regulatory mechanisms controlling Ski and SnoN stability, subcellular localization and expression are altered in hepatocarcinoma cells. GENERAL SIGNIFICANCE This work provides evidence that Ski and SnoN protein regulation is far more complex in normal than in transformed cells, since many of the normal regulatory mechanisms are lost in transformed cells.

Calcium-sensing receptor inhibits TGF-β-signaling by decreasing Smad2 phosphorylation

Calcium‐sensing receptor (CaSR) contributes to maintain homeostatic levels of extracellular calcium. In addition, CaSR controls other cellular activities such as proliferation and migration, particularly in cells not related to extracellular calcium homeostasis, potentially by cross‐talking with parallel signaling pathways. Here we report that CaSR attenuates transforming growth factor‐β (TGF‐β)‐signaling in hepatic C9 cells and in transfected HEK293 cells. Wild type CaSR interferes with TGF‐β‐dependent Smad2 phosphorylation and induces its proteasomal degradation, resulting in a decrease of TGF‐β‐dependent transcriptional activity, whereas an inactivating CaSR mutant does not transduce an inhibitory effect of extracellular calcium on TGF‐β signaling. Attenuation of TGF‐β signaling in response to extracellular calcium is linked to Rab11‐dependent CaSR‐trafficking with the intervention of CaSR carboxyl‐terminal tail. Our data suggest that CaSR might regulate TGF‐β‐dependent cellular responses mediated by TGF‐β signaling inhibition.

Downregulation of SnoN oncoprotein induced by antibiotics anisomycin and puromycin positively regulates transforming growth factor-β signals.

BACKGROUND SnoN and Ski proteins function as Smad transcriptional corepressors and are implicated in the regulation of diverse cellular processes such as proliferation, differentiation and transformation. Transforming growth factor-β (TGF-β) signaling causes SnoN and Ski protein degradation via proteasome with the participation of phosphorylated R-Smad proteins. Intriguingly, the antibiotics anisomycin (ANS) and puromycin (PURO) are also able to downregulate Ski and SnoN proteins via proteasome. METHODS We explored the effects of ANS and PURO on SnoN protein downregulation when the activity of TGF-β signaling was inhibited by using different pharmacological and non-pharmacological approaches, either by using specific TβRI inhibitors, overexpressing the inhibitory Smad7 protein, or knocking-down TβRI receptor or Smad2 by specific shRNAs. The outcome of SnoN and Ski downregulation induced by ANS or PURO on TGF-β signaling was also studied. RESULTS SnoN protein downregulation induced by ANS and PURO did not involve the induction of R-Smad phosphorylation but it was abrogated after TGF-β signaling inhibition; this effect occurred in a cell type-specific manner and independently of protein synthesis inhibition or any other ribotoxic effect. Intriguingly, antibiotics seem to require components of the TGF-β/Smad pathway to downregulate SnoN. In addition, SnoN protein downregulation induced by antibiotics favored gene transcription induced by TGF-β signaling. CONCLUSIONS ANS and PURO require TGF-β/Smad pathway to induce SnoN and Ski protein downregulation independently of inducing R-Smad2 phosphorylation, which facilitates TGF-β signaling. GENERAL SIGNIFICANCE Antibiotic analogs lacking ribotoxic effects are useful as pharmacological tools to study TGF-β signaling by controlling Ski and SnoN protein levels.