Characterization of Novel Derivatives of MBQ-167, an inhibitor of the GTP-binding proteins Rac/Cdc42.

Rac and Cdc42, are homologous GTPases that regulate cell migration, invasion, and cell cycle progression; thus, representing key targets for metastasis therapy. We previously reported on the efficacy of MBQ-167, which blocks both Rac1 and Cdc42 in breast cancer cells and mouse models of metastasis. To identify compounds with increased activity, a panel of MBQ-167 derivatives was synthesized, maintaining its 9-ethyl-3-(1H-1,2,3-triazol-1-yl)-9H-carbazole core. Similar to MBQ-167, MBQ-168 and EHop-097, inhibit activation of Rac and Rac1B splice variant and breast cancer cell viability, and induce apoptosis. MBQ-167 and MBQ-168 inhibit Rac and Cdc42 by interfering with guanine nucleotide binding, and MBQ-168 is a more effective inhibitor of PAK (1,2,3) activation. EHop-097 acts via a different mechanism by inhibiting the interaction of the guanine nucleotide exchange factor (GEF) Vav with Rac. MBQ-168 and EHop-097 inhibit metastatic breast cancer cell migration, and MBQ-168 promotes loss of cancer cell polarity to result in disorganization of the actin cytoskeleton and detachment from the substratum. In lung cancer cells, MBQ-168 is more effective than MBQ-167 or EHop-097 at reducing ruffle formation in response to EGF. Comparable to MBQ-167, MBQ-168 significantly inhibits HER2+ tumor growth and metastasis to lung, liver, and spleen. Both MBQ-167 and MBQ-168 inhibit the cytochrome P450 (CYP) enzymes 3A4, 2C9, and 2C19. However, MBQ-168 is ~10X less potent than MBQ-167 at inhibiting CYP3A4, thus demonstrating its utility in relevant combination therapies. In conclusion, the MBQ-167 derivatives MBQ-168 and EHop-097 are additional promising anti metastatic cancer compounds with similar and distinct mechanisms.

Rac inhibition as a novel therapeutic strategy for EGFR/HER2 targeted therapy resistant breast cancer.

BACKGROUND: Even though targeted therapies are available for cancers expressing oncogenic epidermal growth receptor (EGFR) and (or) human EGFR2 (HER2), acquired or intrinsic resistance often confounds therapy success. Common mechanisms of therapy resistance involve activating receptor point mutations and (or) upregulation of signaling downstream of EGFR/HER2 to Akt and (or) mitogen activated protein kinase (MAPK) pathways. However, additional pathways of resistance may exist thus, confounding successful therapy. METHODS: To determine novel mechanisms of EGFR/HER2 therapy resistance in breast cancer, gefitinib or lapatinib resistant variants were created from SKBR3 breast cancer cells. Syngenic therapy sensitive and resistant SKBR3 variants were characterized for mechanisms of resistance by mammosphere assays, viability assays, and western blotting for total and phospho proteins. RESULTS: Gefitinib and lapatinib treatments reduced mammosphere formation in the sensitive cells, but not in the therapy resistant variants, indicating enhanced mesenchymal and cancer stem cell-like characteristics in therapy resistant cells. The therapy resistant variants did not show significant changes in known therapy resistant pathways of AKT and MAPK activities downstream of EGFR/HER2. However, these cells exhibited elevated expression and activation of the small GTPase Rac, which is a pivotal intermediate of GFR signaling in EMT and metastasis. Therefore, the potential of the Rac inhibitors EHop-016 and MBQ-167 to overcome therapy resistance was tested, and found to inhibit viability and induce apoptosis of therapy resistant cells. CONCLUSIONS: Rac inhibition may represent a viable strategy for treatment of EGFR/HER2 targeted therapy resistant breast cancer.

Gßγ recruits and activates P-Rex1 via two independent binding interfaces.

Gßγ marks the inner side of the plasma membrane where chemotactic GPCRs activate Rac to lead the assembly of actin filaments that push the cell to move forward. Upon dissociation from heterotrimeric Gi, Gßγ recruits and activates P-Rex1, a Rac guanine nucleotide exchange factor (RacGEF). This cytosolic chemotactic effector is kept inactive by intramolecular interactions. The mechanism by which Gßγ stimulates P-Rex1 has been debated. We hypothesized that Gßγ activates P-Rex1 by a two-step mechanism based on independent interaction interfaces to recruit and unroll this RacGEF. Using pulldown assays, we found that Gßγ binds P-Rex1-DH/PH as well as PDZ-PDZ domains. These domains and the DEP-DEP tandem interact among them and dissociate upon binding with Gßγ, arguing for a stimulatory allosteric effect. In addition, P-Rex1 catalytic activity is inhibited by its C-terminal domain. To discern P-Rex1 recruitment from activation, we studied Q-Rhox, a synthetic RhoGEF having the PDZ-RhoGEF catalytic DH/PH module, insensitive to Gßγ, swapped into P-Rex1. Gßγ recruited Q-Rhox to the plasma membrane, indicating that Gßγ/PDZ-PDZ interaction interface plays a role on P-Rex1 recruitment. In conclusion, we reconcile previous findings and propose a mechanistic model of P-Rex1 activation; accordingly, Gßγ recruits P-Rex1 via the Gßγ/PDZ-PDZ interface followed by a second contact involving the Gßγ/DH/PH interface to unleash P-Rex1 RacGEF activity at the plasma membrane.

Identifying a novel 5-gene signature predicting clinical outcomes in acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is the most common type of acute leukemia and biologically heterogeneous diseases with poor prognosis. Thus, we aimed to identify prognostic markers to effectively predict the prognosis of AML patients and eventually guide treatment. METHODS: Prognosis-associated genes were determined by Kaplan-Meier and multivariate analyses using the expression and clinical data of 173 AML patients from The Cancer Genome Atlas database and validated in an independent Oregon Health and Science University dataset. A prognostic risk score was computed based on a linear combination of 5-gene expression levels using the regression coefficients derived from the multivariate logistic regression model. The classification of AML was established by unsupervised hierarchical clustering of CALCRL, DOCK1, PLA2G4A, FCHO2 and LRCH4 expression levels. RESULTS: High FCHO2 and LRCH4 expression was related to decreased mortality. While high CALCRL, DOCK1, PLA2G4A expression was associated with increased mortality. The risk score was predictive of increased mortality rate in AML patients. Hierarchical clustering analysis of the five genes discovered three clusters of AML patients. The cluster1 AML patients were associated with lower cytogenetics risk than cluster2 or 3 patients, and better prognosis than cluster3 patients (P values < 0.05 for all cases, fisher exact test or log-rank test). CONCLUSION: The gene panel comprising CALCRL, DOCK1, PLA2G4A, FCHO2 and LRCH4 as well as the risk score may offer novel prognostic biomarkers and classification of AML patients to significantly improve outcome prediction.

Immunochip meta-analysis in European and Argentinian populations identifies two novel genetic loci associated with celiac disease.

Celiac disease (CeD) is a common immune-mediated disease of the small intestine that is triggered by exposure to dietary gluten. While the HLA locus plays a major role in disease susceptibility, 39 non-HLA loci were also identified in a study of 24,269 individuals. We now build on this earlier study by adding 4125 additional Caucasian samples including an Argentinian cohort. In doing so, we not only confirm the previous associations, we also identify two novel independent genome-wide significant associations at loci: 12p13.31 and 22q13.1. By applying a genomics approach and differential expression analysis in CeD intestinal biopsies, we prioritize potential causal genes at these novel loci, including LTBR, CYTH4, and RAC2. Nineteen prioritized causal genes are overlapping known drug targets. Pathway enrichment analysis and expression of these genes in CeD biopsies suggest that they have roles in regulating multiple pathways such as the tumor necrosis factor (TNF) mediated signaling pathway and positive regulation of I-κB kinase/NF-κB signaling.

Actin cytoskeleton dynamics in stem cells from autistic individuals.

Several lines of indirect evidence, such as mutations or dysregulated expression of genes related to cytoskeleton, have suggested that cytoskeletal dynamics, a process essential for axons and dendrites development, is compromised in autism spectrum disorders (ASD). However, no study has yet examined whether cytoskeleton dynamics is functionally altered in cells from ASD patients. Here we investigated the regulation of actin cytoskeleton dynamics in stem cells from human exfoliated deciduous teeth (SHEDs) of 13 ASD patients and 8 control individuals by inducing actin filament depolymerization and then measuing their reconstruction upon activation of the RhoGTPases Rac, Cdc42 or RhoA. We observed that stem cells from seven ASD individuals (53%) presented altered dymanics of filament reconstruction, including a patient recently studied by our group whose iPSC-derived neuronal cells show shorten and less arborized neurites. We also report potentially pathogenic genetic variants that might be related to the alterations in actin repolymerization dynamics observed in some patient-derived cells. Our results suggest that, at least for a subgroup of ASD patients, the dynamics of actin polymerization is impaired, which might be ultimately leading to neuronal abnormalities.

Targeting Rac and Cdc42 GTPases in Cancer.

Rac and Cdc42 are small GTPases that have been linked to multiple human cancers and are implicated in epithelial to mesenchymal transition, cell-cycle progression, migration/invasion, tumor growth, angiogenesis, and oncogenic transformation. With the exception of the P29S driver mutation in melanoma, Rac and Cdc42 are not generally mutated in cancer, but are overexpressed (gene amplification and mRNA upregulation) or hyperactivated. Rac and Cdc42 are hyperactivated via signaling through oncogenic cell surface receptors, such as growth factor receptors, which converge on the guanine nucleotide exchange factors that regulate their GDP/GTP exchange. Hence, targeting Rac and Cdc42 represents a promising strategy for precise cancer therapy, as well as for inhibition of bypass signaling that promotes resistance to cell surface receptor-targeted therapies. Therefore, an understanding of the regulatory mechanisms of these pivotal signaling intermediates is key for the development of effective inhibitors. In this review, we focus on the role of Rac and Cdc42 in cancer and summarize the regulatory mechanisms, inhibitory efficacy, and the anticancer potential of Rac- and Cdc42-targeting agents. Cancer Res; 78(12); 3101-11. ©2018 AACR.

Cell adhesion controlled by adhesion G protein-coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo-Dock.

Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gßγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses.

A new nucleocytoplasmic RhoGAP protein contributes to control the pathogenicity of Entamoeba histolytica by regulating EhRacC and EhRacD activity.

Small GTPases are signalling molecules that regulate important cellular processes. GTPases are deactivated by GTPase-activating proteins (GAPs). While human GAPs have been intensively studied, no GAP has yet been characterized in Entamoeba histolytica. In this study, we identified and characterized a novel nucleocytoplasmic RhoGAP in E. histolytica termed EhRhoGAPnc. In silico analyses of the domain structure revealed a previously undescribed peptide region within the carboxy-terminal region of EhRhoGAPnc capable of interacting with phosphatidic acid and phosphatidylinositol 3,5-bisphosphate. The full structural GAP domain showed increase GAP activity compared with the minimum region able to display GAP activity, as analysed both by experimental assays and molecular dynamics simulations. Furthermore, we identified amino acid residues that promote interactions between EhRhoGAPnc and its target GTPases EhRacC and EhRacD. Immunofluorescence studies revealed that EhRhoGAPnc colocalized with EhRacC and EhRacD during uroid formation but not during erythrophagocytosis. Interestingly, during erythrophagocytosis of red blood cells, EhRhoGAPnc colocalized with phosphatidic acid and phosphatidylinositol 3,5-bisphosphate. Overexpression of EhRhoGAPnc in E. histolytica led to inhibition of actin adhesion plate formation, migration, adhesion of E. histolytica to MDCK cells and consequently to an impairment of the cytopathic activity.

RAC3 more than a nuclear receptor coactivator: a key inhibitor of senescence that is downregulated in aging.

Receptor-associated coactivator 3 (RAC3) is a nuclear receptor coactivator usually overexpressed in tumors that exerts oncogenic functions in the cytoplasm and the nucleus. Although as part of its oncogenic actions it was previously identified as an inhibitor of apoptosis and autophagy, its expression is required in order to preserve the pluripotency and embryonic stem cell self-renewal. In this work we investigated its role in cellular senescence. We found that RAC3 overexpression in the nontumoral HEK293 cells inhibits the premature senescence induced by hydrogen peroxide or rapamycin. The mechanism involves not only the inhibition of autophagy early induced by these stimuli in the pathway to senescence, but also the increase in levels and nuclear localization of both the cell cycle suppressors p53/p21 and the longevity promoters FOXO1A, FOXO3A and SIRT1. Furthermore, we found that RAC3 overexpression is required in order to maintain the telomerase activity. In tumoral HeLa cells its activity was inhibited by depletion of RAC3 inducing replicative senescence. Moreover, we demonstrated that in vivo, levels of RAC3 are downregulated in the liver from aged as compared with young rats, whereas the levels of p21 are increased, correlating with the expected senescent cell contents in aged tissues. A similar downregulation of RAC3 was observed in the premature and replicative senescence of human fetal WI-38 cells and premature senescence of hepatocyte HepG2 cell line. Taken together, all these results demonstrate that RAC3 is an inhibitor of senescence whose downregulation in aged individuals could be probably a tumor suppressor mechanism, avoiding the clonal expansion of risky old cells having damaged DNA.

Vascular injury in diabetic db/db mice is ameliorated by atorvastatin: role of Rac1/2-sensitive Nox-dependent pathways.

Oxidative stress [increased bioavailability of reactive oxygen species (ROS)] plays a role in the endothelial dysfunction and vascular inflammation, which underlie vascular damage in diabetes. Statins are cholesterol-lowering drugs that are vasoprotective in diabetes through unknown mechanisms. We tested the hypothesis that atorvastatin decreases NADPH oxidase (Nox)-derived ROS generation and associated vascular injury in diabetes. Lepr(db)/Lepr(db) (db/db) mice, a model of Type 2 diabetes and control Lepr(db)/Lepr(+) (db/+) mice were administered atorvastatin (10 mg/kg per day, 2 weeks). Atorvastatin improved glucose tolerance in db/db mice. Systemic and vascular oxidative stress in db/db mice, characterized by increased plasma TBARS (thiobarbituric acid-reactive substances) levels and exaggerated vascular Nox-derived ROS generation respectively, were inhibited by atorvastatin. Cytosol-to-membrane translocation of the Nox regulatory subunit p47(phox) and the small GTPase Rac1/2 was increased in vessels from db/db mice compared with db/+ mice, an effect blunted by atorvastatin. The increase in vascular Nox1/2/4 expression and increased phosphorylation of redox-sensitive mitogen-activated protein kinases (MAPKs) was abrogated by atorvastatin in db/db mice. Pro-inflammatory signalling (decreased IκB-α and increased NF-κB p50 expression, increased NF-κB p65 phosphorylation) and associated vascular inflammation [vascular cell adhesion molecule-1 (VCAM-1) expression and vascular monocyte adhesion], which were increased in aortas of db/db mice, were blunted by atorvastatin. Impaired acetylcholine (Ach)- and insulin (INS)-induced vasorelaxation in db/db mice was normalized by atorvastatin. Our results demonstrate that, in diabetic mice, atorvastatin decreases vascular oxidative stress and inflammation and ameliorates vascular injury through processes involving decreased activation of Rac1/2 and Nox. These findings elucidate redox-sensitive and Rac1/2-dependent mechanisms whereby statins protect against vascular injury in diabetes.

Defective Wound-healing in Aging Gingival Tissue.

Aging may negatively affect gingival wound-healing. However, little is known about the mechanisms underlying this phenomenon. The present study examined the cellular responses associated with gingival wound-healing in aging. Primary cultures of human gingival fibroblasts were obtained from healthy young and aged donors for the analysis of cell proliferation, cell invasion, myofibroblastic differentiation, and collagen gel remodeling. Serum from young and old rats was used to stimulate cell migration. Gingival repair was evaluated in Sprague-Dawley rats of different ages. Data were analyzed by the Mann-Whitney and Kruskal-Wallis tests, with a p value of .05. Fibroblasts from aged donors showed a significant decrease in cell proliferation, migration, Rac activation, and collagen remodeling when compared with young fibroblasts. Serum from young rats induced higher cell migration when compared with serum from old rats. After TGF-beta1 stimulation, both young and old fibroblasts demonstrated increased levels of alpha-SMA. However, alpha-SMA was incorporated into actin stress fibers in young but not in old fibroblasts. After 7 days of repair, a significant delay in gingival wound-healing was observed in old rats. The present study suggests that cell migration, myofibroblastic differentiation, collagen gel remodeling, and proliferation are decreased in aged fibroblasts. In addition, altered cell migration in wound-healing may be attributable not only to cellular defects but also to changes in serum factors associated with the senescence process.

ß3-chimaerin, a novel member of the chimaerin Rac-GAP family.

Chimaerins are a family of diacylglycerol- and phorbol ester-regulated GTPase activating proteins (GAPs) for the small G-protein Rac. Extensive evidence indicates that these proteins play important roles in development, axon guidance, metabolism, cell motility, and T cell activation. Four isoforms have been reported to-date, which are products of CHN1 (α1- and α2-chimaerins) and CHN2 (ß1- and ß2-chimaerins) genes. Although these gene products are assumed to be generated by alternative splicing, bioinformatics analysis of the CHN2 gene revealed that ß1- and ß2-chimaerins are the products of alternative transcription start sites (TSSs) in different promoter regions. Furthermore, we found an additional TSS in CHN2 gene that leads to a novel product, which we named ß3-chimaerin. Expression profile analysis revealed predominantly low levels for the ß3-chimaerin transcript, with higher expression levels in epididymis, plasma blood leucocytes, spleen, thymus, as well as various areas of the brain. In addition to the prototypical SH2, C1, and Rac-GAP domains, ß3-chimaerin has a unique N-terminal domain. Studies in cells established that ß3-chimaerin has Rac-GAP activity and is responsive to phorbol esters. The enhanced responsiveness of ß3-chimaerin for phorbol ester-induced translocation relative to ß2-chimaerin suggests differential ligand accessibility to the C1 domain.

Activation of Akt pathway by transcription-independent mechanisms of retinoic acid promotes survival and invasion in lung cancer cells.

BACKGROUND: All-trans retinoic acid (ATRA) is currently being used in clinical trials for cancer treatment. The use of ATRA is limited because some cancers, such as lung cancer, show resistance to treatment. However, little is known about the molecular mechanisms that regulate resistance to ATRA treatment. Akt is a kinase that plays a key role in cell survival and cell invasion. Akt is often activated in lung cancer, suggesting its participation in resistance to chemotherapy. In this study, we explored the hypothesis that activation of the Akt pathway promotes resistance to ATRA treatment at the inhibition of cell survival and invasion in lung cancer. We aimed to provide guidelines for the proper use of ATRA in clinical trials and to elucidate basic biological mechanisms of resistance. RESULTS: We performed experiments using the A549 human lung adenocarcinoma cell line. We found that ATRA treatment promotes PI3k-Akt pathway activation through transcription-independent mechanisms. Interestingly, ATRA treatment induces the translocation of RARα to the plasma membrane, where it colocalizes with Akt. Immunoprecipitation assays showed that ATRA promotes Akt activation mediated by RARα-Akt interaction. Activation of the PI3k-Akt pathway by ATRA promotes invasion through Rac-GTPase, whereas pretreatment with 15e (PI3k inhibitor) or over-expression of the inactive form of Akt blocks ATRA-induced invasion. We also found that treatment with ATRA induces cell survival, which is inhibited by 15e or over-expression of an inactive form of Akt, through a subsequent increase in the levels of the active form of caspase-3. Finally, we showed that over-expression of the active form of Akt significantly decreases expression levels of the tumor suppressors RARß2 and p53. In contrast, over-expression of the inactive form of Akt restores RARß2 expression in cells treated with ATRA, indicating that activation of the PI3k-Akt pathway inhibits the expression of ATRA target genes. CONCLUSION: Our results demonstrate that rapid activation of Akt blocks transcription-dependent mechanism of ATRA, promotes invasion and cell survival and confers resistance to retinoic acid treatment in lung cancer cells. These findings provide an incentive for the design and clinical testing of treatment regimens that combine ATRA and PI3k inhibitors for lung cancer treatment.

Nuclear receptor coactivator RAC3 inhibits autophagy.

RAC3 is an oncogene naturally overexpressed in several tumors. Besides its role as coactivator, it can exert several protumoral cytoplasmic actions. Autophagy was found to act either as a tumor suppressor during the early stages of tumor development, or as a protector of the tumor cell in later stages under hypoxic conditions. We found that RAC3 overexpression inhibits autophagy when induced by starvation or rapamycin and involves RAC3 nuclear translocation-dependent and -independent mechanisms. Moreover, hypoxia inhibits the RAC3 gene expression leading to the autophagy process, allowing tumor cells to survive until angiogenesis occurs. The interplay between RAC3, hypoxia, and autophagy could be an important mechanism for tumor progression and a good target for a future anticancer therapy.

Cyclin D1 is a NF-κB corepressor.

NF-κB regulates the expression of Cyclin D1 (CD1), while RAC3 is an NF-κB coactivator that enhances its transcriptional activity. In this work, we investigated the regulatory role of CD1 on NF-κB activity. We found that CD1 inhibits NF-κB transcriptional activity through a corepressor function that can be reverted by over-expressing RAC3. In both, tumoral and non-tumoral cells, the expression pattern of RAC3 and CD1 is regulated by the cell cycle, showing a gap between the maximal expression levels of each protein. The individual increase, by transfection, of either CD1 or RAC3 enhances cell proliferation. However the simultaneous and constitutive over-expression of both proteins has an inhibitory effect. Our results suggest that the relative amounts of CD1 and RAC3, and the timing of expression of these oncogenes could tilt the balance of tumor cell proliferation in response to external signals.

Involvement of hnRNP A1 in the matrix metalloprotease-3-dependent regulation of Rac1 pre-mRNA splicing.

Rac1b is an alternatively spliced isoform of the small GTPase Rac1 that includes the 57-nucleotide exon 3b. Rac1b was originally identified through its over-expression in breast and colorectal cancer cells, and has subsequently been implicated as a key player in a number of different oncogenic signaling pathways, including tumorigenic transformation of mammary epithelial cells exposed to matrix metalloproteinase-3 (MMP-3). Although many of the cellular consequences of Rac1b activity have been recently described, the molecular mechanism by which MMP-3 treatment leads to Rac1b induction has not been defined. Here we use proteomic methods to identify heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a factor involved in Rac1 splicing regulation. We find that hnRNP A1 binds to Rac1 exon 3b in mouse mammary epithelial cells, repressing its inclusion into mature mRNA. We also find that exposure of cells to MMP-3 leads to release of hnRNP A1 from exon 3b and the consequent generation of Rac1b. Finally, we analyze normal breast tissue and breast cancer biopsies, and identify an inverse correlation between expression of hnRNP A1 and Rac1b, suggesting the existence of this regulatory axis in vivo. These results provide new insights on how extracellular signals regulate alternative splicing, contributing to cellular transformation and development of breast cancer.

Fibrillar amyloid-ß1-42 modifies actin organization affecting the cofilin phosphorylation state: a role for Rac1/cdc42 effector proteins and the slingshot phosphatase.

The neuronal cytoskeleton regulates numerous processes that occur in normal homeostasis. Under pathological conditions such as those of Alzheimer's disease (AD), major alterations in cytoskeleton organization have been observed and changes in both microtubules and actin filaments have been reported. Many neurodegenerative consequences of AD are linked to the production and accumulation of amyloid peptides (Aß) and their oligomers, produced from the internal cleavage of the amyloid-ß protein precursor. We previously reported that fibrillar Aß1-42 (fAß) treatment of hippocampal neurons induced an increase in Rac1 and Cdc42 activities linking fAß effects with changes in actin dynamics. Here we show fAß-induces increased activity of PAK1 and cyclin-dependent kinase 5, and that p21-activated kinase (PAK1) activation targets the LIMK1-cofilin signaling pathway. Increased cofilin dephosphorylation under conditions of enhanced LIM-Kinase 1 (LIMK1) activity suggests that fAß co-stimulates bifurcating pathways impacting cofilin phosphorylation. Overexpression of slingshot (SSH) prevents the augment of F-actin induced by fAß after 24 h, suggesting that fAß-induced changes in actin assembly involve both LIMK1 and SSH. These results suggest that fAb may alter the PAK1/LIMK1/cofilin axis and therefore actin organization in AD.

Simvastatin alters fibroblastic cell responses involved in tissue repair.

BACKGROUND AND OBJECTIVE: Statins have been used to control hypercholesterolemia. However, these drugs also exert pleiotropic effects that include the modulation of inflammation and cell signaling. The present study has analyzed the effects of simvastatin on several cell responses involved in tissue repair, including cell adhesion, cell migration and invasion, actin cytoskeleton remodeling and cell viability. MATERIAL AND METHODS: Primary cultures of gingival fibroblasts were stimulated with simvastatin. Cell adhesion was evaluated using a colorimetric assay. Cell spreading was evaluated microscopically. Cell migration and invasion were assessed using a scratch wound-healing assay and a bicameral cell culture system, respectively. Changes in actin cytoskeleton and focal adhesion assembly were evaluated through immunofluorescence for actin, vinculin and active ß1 integrin. Rac activation was evaluated by means of a pull-down assay. Cell viability was assessed using a colorimetric assay that determines mitochondrial functionality. Data analysis was performed using the Mann-Whitney U-test. RESULTS: Simvastatin diminished cell adhesion and spreading over a fibronectin matrix. It also altered the closure of scratch wounds induced on cell monolayers and cell invasion through a Transwell system. Simvastatin-treated cells displayed an altered lamellipodia with poorly developed focal adhesion contacts and reduced levels of ß1 integrin activation. During cell spreading, simvastatin diminished Rac activation. CONCLUSION: The present study shows that simvastatin may alter cell migration by disrupting the cell signaling networks that regulate the actin cytoskeleton dynamics. This mechanism may affect the response of gingival mesenchymal cells during wound healing.

Cholesterol modulates the rate and mechanism of acetylcholine receptor internalization.

Stability of the nicotinic acetylcholine receptor (AChR) at the cell surface is key to the correct functioning of the cholinergic synapse. Cholesterol (Chol) is necessary for homeostasis of AChR levels at the plasmalemma and for ion translocation. Here we characterize the endocytic pathway followed by muscle-type AChR in Chol-depleted cells (Chol(-)). Under such conditions, the AChR is internalized by a ligand-, clathrin-, and dynamin-independent mechanism. Expression of a dominant negative form of the small GTPase Rac1, Rac1N17, abolishes receptor endocytosis. Unlike the endocytic pathway in control CHO cells (1), accelerated AChR internalization proceeds even upon disruption of the actin cytoskeleton. Under Chol(-) conditions, AChR internalization is furthermore found to require the activity of Arf6 and its effectors Rac1 and phospholipase D. The Arf6-dependent mechanism may constitute the default endocytic pathway followed by the AChR in the absence of external ligands, membrane Chol levels acting as a key homeostatic regulator of cell surface receptor levels.