Cofilin Phosphorylation Research Articles

Hippocampal LIMK1-mediated Structural Synaptic Plasticity in Neurobehavioral Deficits Induced by a Low-dose Heavy Metal Mixture.

Humans are commonly exposed to the representative neurotoxic heavy metals lead (Pb), cadmium (Cd), and mercury (Hg). These three substances can be detected simultaneously in the blood of the general population. We have previously shown that a low-dose mixture of these heavy metals induces rat learning and memory impairment at human exposure levels, but the pathogenic mechanism is still unclear. LIM kinase 1 (LIMK1) plays a critical role in orchestrating synaptic plasticity during brain function and dysfunction. Hence, we investigated the role of LIMK1 activity in low-dose heavy metal mixture-induced neurobehavioral deficits and structural synaptic plasticity disorders. Our results showed that heavy metal mixture exposure altered rat fear responses and spatial learning at general population exposure levels and that these alterations were accompanied by downregulation of LIMK1 phosphorylation and structural synaptic plasticity dysfunction in rat hippocampal tissues and cultured hippocampal neurons. In addition, upregulation of LIMK1 phosphorylation attenuated heavy metal mixture-induced structural synaptic plasticity, dendritic actin dynamics, and cofilin phosphorylation damage. The potent LIMK1 inhibitor BMS-5 yielded similar results induced by heavy metal mixture exposure and aggravated these impairments. Our findings demonstrate that LIMK1 plays a crucial role in neurobehavioral deficits induced by low-dose heavy metal mixture exposure by suppressing structural synaptic plasticity.

Inhibition of airway smooth muscle contraction and proliferation by LIM kinase inhibitor, LIMKi3

PurposeCurrent medical treatment for asthma aims to inhibit airway smooth muscle (ASM) contraction and proliferation, however, the efficacy of available treatment options is unsatisfactory. Therefore, we explored the effect of LIM domain kinase (LIMK) inhibitor - LIMKi3, on ASM to improve the understanding of ASM contraction and proliferation mechanisms, and to investigate new therapeutic targets. Materials and methodsAsthma model was induced in rats by intraperitoneal injection of ovalbumin. Using phospho-specific antibodies, we examined LIMK, phosphorylated LIMK, cofilin and phosphorylated cofilin. ASM contraction was studied in organ bath experiments. ASM cells proliferation was studied with cell counting kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) assays. ResultsImmunofluorescence indicated that LIMKs are expressed in ASM tissues. Western blot revealed that LIMK1 and phospho-cofilin were significantly elevated in asthma ASM tissues. The LIMK inhibitor, LIMKi3 (1 ​μM) could reduce cofilin phosphorylation and therefore inhibit contraction of ASM tissues, and induce actin filament breakdown as well as cell proliferation reduction in cultured human ASM cells. ConclusionsASM contraction and proliferation in asthma may underlie the effects of LIMKs. Small molecule LIMK inhibitor, LIMKi3, might be a potential therapeutic strategy for asthma.

Lysophosphatidylinositol Promotes Chemotaxis and Cytokine Synthesis in Mast Cells with Differential Participation of GPR55 and CB2 Receptors

Mast cells (MCs) are the main participants in the control of immune reactions associated with inflammation, allergies, defense against pathogens, and tumor growth. Bioactive lipids are lipophilic compounds able to modulate MC activation. Here, we explored some of the effects of the bioactive lipid lysophosphatidylinositol (LPI) on MCs. Utilizing murine bone marrow-derived mast cells (BMMCs), we found that LPI did not cause degranulation, but slightly increased FcεRI-dependent β-hexosaminidase release. However, LPI induced strong chemotaxis together with changes in LIM kinase (LIMK) and cofilin phosphorylation. LPI also promoted modifications to actin cytoskeleton dynamics that were detected by an increase in cell size and interruptions in the continuity of the cortical actin ring. The chemotaxis and cortical actin ring changes were dependent on GPR55 receptor activation, since the specific agonist O1602 mimicked the effects of LPI and the selective antagonist ML193 prevented them. The LPI and O1602-dependent stimulation of BMMC also led to VEGF, TNF, IL-1α, and IL-1β mRNA accumulation, but, in contrast with chemotaxis-related processes, the effects on cytokine transcription were dependent on GPR55 and cannabinoid (CB) 2 receptors, since they were sensitive to ML193 and to the specific CB2 receptor antagonist AM630. Remarkably, GPR55-dependent BMMC chemotaxis was observed towards conditioned media from distinct mouse and human cancer cells. Our data suggest that LPI induces the chemotaxis of MCs and leads to cytokine production in MC in vitro with the differential participation of GPR55 and CB2 receptors. These effects could play a significant role in the recruitment of MCs to tumors and the production of MC-derived pro-angiogenic factors in the tumor microenvironment.

The Antiviral Compound PSP Inhibits HIV-1 Entry via PKR-Dependent Activation in Monocytic Cells.

Actin depolymerization factor (ADF) cofilin-1 is a key cytoskeleton component that serves to lessen cortical actin. HIV-1 manipulates cofilin-1 regulation as a pre- and post-entry requisite. Disruption of ADF signaling is associated with denial of entry. The unfolded protein response (UPR) marker Inositol-Requiring Enzyme-1α (IRE1α) and interferon-induced protein (IFN-IP) double-stranded RNA- activated protein kinase (PKR) are reported to overlap with actin components. In our published findings, Coriolus versicolor bioactive extract polysaccharide peptide (PSP) has demonstrated anti-HIV replicative properties in THP1 monocytic cells. However, its involvement towards viral infectivity has not been elucidated before. In the present study, we examined the roles of PKR and IRE1α in cofilin-1 phosphorylation and its HIV-1 restrictive roles in THP1. HIV-1 p24 antigen was measured through infected supernatant to determine PSP's restrictive potential. Quantitative proteomics was performed to analyze cytoskeletal and UPR regulators. PKR, IRE1α, and cofilin-1 biomarkers were measured through immunoblots. Validation of key proteome markers was done through RT-qPCR. PKR/IRE1α inhibitors were used to validate viral entry and cofilin-1 phosphorylation through Western blots. Our findings show that PSP treatment before infection leads to an overall lower infectivity. Additionally, PKR and IRE1α show to be key regulators in cofilin-1 phosphorylation and viral restriction.

The slingshot phosphatase 2 is required for acrosome biogenesis during spermatogenesis in mice.

The acrosome is a membranous organelle positioned in the anterior portion of the sperm head and is essential for male fertility. Acrosome biogenesis requires the dynamic cytoskeletal shuttling of vesicles towards nascent acrosome which is regulated by a series of accessory proteins. However, much remains unknown about the molecular basis underlying this process. Here, we generated Ssh2 knock-out (KO) mice and HA-tagged Ssh2 knock-in (KI) mice to define the functions of Slingshot phosphatase 2 (SSH2) in spermatogenesis and demonstrated that as a regulator of actin remodeling, SSH2 is essential for acrosome biogenesis and male fertility. In Ssh2 KO males, spermatogenesis was arrested at the early spermatid stage with increased apoptotic index and the impaired acrosome biogenesis was characterized by defective transport/fusion of proacrosomal vesicles. Moreover, disorganized F-actin structures accompanied by excessive phosphorylation of COFILIN were observed in the testes of Ssh2 KO mice. Collectively, our data reveal a modulatory role for SSH2 in acrosome biogenesis through COFILIN-mediated actin remodeling and the indispensability of this phosphatase in male fertility in mice.

LIM Kinases, LIMK1 and LIMK2, Are Crucial Node Actors of the Cell Fate: Molecular to Pathological Features

LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) are serine/threonine and tyrosine kinases and the only two members of the LIM kinase family. They play a crucial role in the regulation of cytoskeleton dynamics by controlling actin filaments and microtubule turnover, especially through the phosphorylation of cofilin, an actin depolymerising factor. Thus, they are involved in many biological processes, such as cell cycle, cell migration, and neuronal differentiation. Consequently, they are also part of numerous pathological mechanisms, especially in cancer, where their involvement has been reported for a few years and has led to the development of a wide range of inhibitors. LIMK1 and LIMK2 are known to be part of the Rho family GTPase signal transduction pathways, but many more partners have been discovered over the decades, and both LIMKs are suspected to be part of an extended and various range of regulation pathways. In this review, we propose to consider the different molecular mechanisms involving LIM kinases and their associated signalling pathways, and to offer a better understanding of their variety of actions within the physiology and physiopathology of the cell.

RCAS1 increases cell morphological changes in murine fibroblasts by reducing p38 phosphorylation.

Receptor‑binding cancer antigen expressed on SiSo cells (RCAS1) is a tumor‑associated antigen that is expressed in a number of human malignancies. RCAS1 acts as a ligand for a putative RCAS1 receptor that is present on various human cells including T and B lymphocytes and natural killer cells, in which it induces cell growth inhibition and apoptosis. It has been suggested that RCAS1 might serve an important role in tumor cell evasion from the host immune system. In fact, RCAS1 expression is related to malignant characteristics including tumor size, invasion depth, clinical stage and poor overall survival. The authors previously established doxycycline‑induced RCAS1 overexpression murine fibroblast L cells to analyze the biological functions of RCAS1 and reported that its expression inhibited cell cycle progression via the downregulation of cyclin D3, which subsequently induced apoptosis. Additionally, it was found that RCAS1 expression induced cell morphological changes prior to caspase‑mediated apoptosis. Thus, the present study examined signaling pathways associated with changes in cell morphology that were induced by RCAS1 expression. The data showed that increased RCAS1 expression caused a reduction in actin stress fibers and decreased cofilin phosphorylation. Recent studies have shown that p38 signaling regulates actin polymerization. The data the present study showed that increased RCAS1 expression significantly decreased p38 phosphorylation.

Reelin regulates the migration of late-born hippocampal CA1 neurons via cofilin phosphorylation

Reelin, a large secreted glycoprotein, plays an important role in neuronal migration during brain development. The C-terminal region (CTR) of Reelin is involved in the efficient activation of downstream signaling and its loss leads to abnormal hippocampal layer formation. However, the molecular mechanism by which Reelin CTR regulates hippocampal development remains unknown. Here, we showed that the migration of late-born, but not early-born, neurons is impaired in the knock-in mice in which Reelin CTR is deleted (ΔC-KI mice). The phosphorylation of cofilin, an actin-depolymerizing protein, was remarkably decreased in the hippocampus of the ΔC-KI mice. Exogenous expression of pseudo-phosphorylated cofilin rescued the ectopic positioning of neurons in the hippocampus of ΔC-KI mice. These results suggest that Reelin CTR is required for the migration of late-born neurons in the hippocampus and that this event involves appropriate phosphorylation of cofilin.

Involvement of Paired Immunoglobulin-Like Receptor B in Cognitive Dysfunction Through Hippocampal-Dependent Synaptic Plasticity Impairments in Mice Subjected to Chronic Sleep Restriction

Sleep loss is often associated with cognitive dysfunction. Alterations in the structure and function of synapses in the hippocampus are thought to underlie memory storage. Paired immunoglobulin-like receptor B (PirB) plays a negative role in various neurological diseases by inhibiting axon regeneration and synaptic plasticity. However, the contributions of PirB to the mechanisms underlying the changes in synaptic plasticity after sleep loss that ultimately promote deficits in cognitive function have not been well elucidated. Here, we showed that chronic sleep restriction (CSR) mice displayed cognitive impairment and synaptic deficits accompanied by upregulation of PirB expression in the hippocampus. Mechanistically, PirB caused the dysregulation of actin through the RhoA/ROCK2/LIMK1/cofilin signalling pathway, leading to abnormal structural and functional plasticity, which in turn resulted in cognitive dysfunction. PirB knockdown alleviated synaptic deficits and cognitive impairment after CSR by inhibiting the RhoA/ROCK2/LIMK1/cofilin signalling pathway. Moreover, we found that fasudil, a widely used ROCK2 inhibitor, could mimic the beneficial effect of PirB knockdown and ameliorate synaptic deficits and cognitive impairment, further demonstrating that PirB induced cognitive dysfunction after CSR via the RhoA/ROCK2/LIMK1/cofilin signalling pathway. Our study sheds new light on the role of PirB as an important mediator in modulating the dysfunction of synaptic plasticity and cognitive function via the RhoA/ROCK2/LIMK1/cofilin signalling pathway, which indicated that hippocampal PirB is a promising therapeutic target for counteracting cognitive impairment after CSR.Graphical This illustration depicts the signalling pathway by PirB in mediating cognitive impairment and synaptic deficits in CSR mice. In the hippocampus of CSR mice, the expression level of PirB was significantly increased. In addition, CSR increases RhoA and ROCK2 levels and reduces levels of both LIMK1 and cofilin phosphorylation. PirB knockdown reverses cognitive impairment and synaptic plasticity disorders caused by CSR through the RhoA/ROCK2/LIMK1/cofilin signalling pathway

Preclinical Evaluation of a Novel Small Molecule Inhibitor of LIM Kinases (LIMK) CEL_Amide in Philadelphia-Chromosome Positive (BCR::ABL+) Acute Lymphoblastic Leukemia (ALL).

Ph+ (BCR::ABL+) B-ALL was considered to be high risk, but recent advances in BCR::ABL-targeting TKIs has shown improved outcomes in combination with backbone chemotherapy. Nevertheless, new treatment strategies are needed, including approaches without chemotherapy for elderly patients. LIMK1/2 acts downstream from various signaling pathways, which modifies cytoskeleton dynamics via phosphorylation of cofilin. Upstream of LIMK1/2, ROCK is constitutively activated by BCR::ABL, and upon activation, ROCK leads to the phosphorylation of LIMK1/2, resulting in the inactivation of cofilin by its phosphorylation and subsequently abrogating its apoptosis-promoting activity. Here, we demonstrate the anti-leukemic effects of a novel LIMK1/2 inhibitor (LIMKi) CEL_Amide in vitro and in vivo for BCR::ABL-driven B-ALL. The IC50 value of CEL_Amide was ≤1000 nM in BCR::ABL+ TOM-1 and BV-173 cells and induced dose-dependent apoptosis and cell cycle arrest in these cell lines. LIMK1/2 were expressed in BCR::ABL+ cell lines and patient cells and LIMKi treatment decreased LIMK1 protein expression, whereas LIMK2 expression was unaffected. As expected, CEL_Amide exposure caused specific activating downstream dephosphorylation of cofilin in cell lines and primary cells. Combination experiments with CEL_Amide and BCR::ABL TKIs imatinib, dasatinib, nilotinib, and ponatinib were synergistic for the treatment of both TOM-1 and BV-173 cells. CDKN2Ako/BCR::ABL1+ B-ALL cells were transplanted in mice, which were treated with combinations of CEL_Amide and nilotinib or ponatinib, which significantly prolonged their survival. Altogether, the LIMKi CEL_Amide yields activity in Ph+ ALL models when combined with BCR::ABL-targeting TKIs, showing promising synergy that warrants further investigation.

Programmable integrin and N-cadherin adhesive interactions modulate mechanosensing of mesenchymal stem cells by cofilin phosphorylation

During mesenchymal development, the sources of mechanical forces transduced by cells transition over time from predominantly cell-cell interactions to predominantly cell-extracellular matrix (ECM) interactions. Transduction of the associated mechanical signals is critical for development, but how these signals converge to regulate human mesenchymal stem cells (hMSCs) mechanosensing is not fully understood, in part because time-evolving mechanical signals cannot readily be presented in vitro. Here, we established a DNA-driven cell culture platform that could be programmed to present the RGD peptide from fibronectin, mimicking cell-ECM interactions, and the HAVDI peptide from N-cadherin, mimicking cell-cell interactions, through DNA hybridization and toehold-mediated strand displacement reactions. The platform could be programmed to mimic the evolving cell-ECM and cell-cell interactions during mesenchymal development. We applied this platform to reveal that RGD/integrin ligation promoted cofilin phosphorylation, while HAVDI/N-cadherin ligation inhibited cofilin phosphorylation. Cofilin phosphorylation upregulated perinuclear apical actin fibers, which deformed the nucleus and thereby induced YAP nuclear localization in hMSCs, resulting in subsequent osteogenic differentiation. Our programmable culture platform is broadly applicable to the study of dynamic, integrated mechanobiological signals in development, healing, and tissue engineering.

Fluid shear stress promotes periodontal ligament cells proliferation via p38-AMOT-YAP.

Periodontal ligament (PDL) cells are a promising tool for periodontal regeneration therapy. Achieving a sufficient number of PDL cells is essential to PDL regeneration. In our study, appropriate flow shear stress (FSS, 1-6 dyn/cm2) promotes the proliferation of PDL cells. FSS remodels cytoskeleton and focal adhesion in a duration-dependent manner. FSS induces PDL cells to form the actin cap within 10min, flattens the nuclei, and increases the nuclear pore size, which promotes nuclear translocation of Yes-associated protein (YAP). FSS activates p38, which plays a dual function in YAP regulation. p38 regulates the phosphorylation of Akt and cofilin, as well as induced F-actin polymerization to induce YAP activity. In addition, p38 inhibits pLATS and consecutively regulates angiomotin (AMOT) and YAP phosphorylation. AMOT competitively binds to F-actin and YAP to participate in FSS-mediated YAP nuclear translocation and cell proliferation. Taken collectively, our results provide mechanistic insights into the role of p38-AMOT-YAP in FSS-mediated PDL cells proliferation and indicate potential applications in dental regenerative medicine.

The UV-induced uptake of melanosome by skin keratinocyte is triggered by α7 nicotinic acetylcholine receptor-mediated phagocytosis.

The melanosome is an organelle that produces melanin for skin pigmentation, which is synthesized by epidermal melanocytes, subsequently transported and internalized by epidermal keratinocytes. Exposure to ultraviolet (UV) from sunlight radiation is a major stimulator of melanosome uptake by keratinocytes. Acetylcholine (ACh) is known to be released by keratinocytes under UV exposure, which regulates melanin production in melanocytes by participating in which has been named as 'skin synapse'. Here, the role of cholinergic molecules, i.e. ACh and α7 nicotinic acetylcholine receptor (nAChR), in regulating melanosome uptake through phagocytosis by keratinocytes was illustrated. In cultured keratinocytes (HaCaT cells), the fluorescent beads at different sizes imitating melanosomes, or melanosomes, were phagocytosed under UV exposure. The UV-induced phagocytosis in keratinocytes was markedly increased by applied ACh, an acetylcholinesterase (AChE) inhibitor or an α7 nAChR agonist. By contrast, the antagonist of α7 nAChR was able to fully block the UV-induced phagocytosis, suggesting the role of α7 nAChR in this event. The intracellular Ca++ mobilization was triggered by UV exposure, accounting for the initiation of phagocytosis. The blockage of UV-mediated Ca++ mobilization, triggered by BAPTA-AM or α7 nAChR antagonist, resulted in a complete termination of phagocytosis. Besides, the phosphorylation of cofilin, as well as expression and activation of RhoA, accounting for phagocytosis was induced by UV exposure: the phosphorylation was blocked by BAPTA-AM or α7 nAChR antagonist. The result suggests that the cholinergic system, especially α7 nAChR, is playing a regulatory role in modulating melanosome uptake in keratinocytes being induced by UV exposure.

RhoA improves cryopreservation of rooster sperm through the Rho/RhoA-associated kinase/cofilin pathway

Cryopreservation of rooster sperm leads to relatively low semen quality due to cytoskeletal damage during the freeze-thawing process. This study aimed to explore how the addition of RhoA recombinant protein affected the viability and subcellular structure of rooster sperm after freeze-thawing and elucidated the molecular mechanisms of sperm cryopreservation. Semen quality and acrosome integrity testing revealed that the addition of 0.5 μg/mL RhoA recombinant protein to the cryoprotectant fluid significantly increased sperm motility, survival rate, linearity, straight-line velocity, and acrosome integrity after freeze-thawing (P < 0.05). Ultrastructure analysis of cryopreserved sperm showed structural damage to the sperm plasma membrane, nuclear membrane, and tail. However, compared to the control, these structural changes were reduced upon the addition of RhoA recombinant protein to the cryoprotective fluid (P < 0.05). Western blotting revealed that the expression of Rho/RhoA-associated kinase and p-cofilin was increased, and cofilin expression was decreased after sperm cryopreservation with recombinant RhoA protein. Treatment with Y-27632, a ROCK antagonist, suppressed ROCK and p-cofilin expression and decreased semen quality, acrosome integrity, and ultrastructure integrity. In summary, we have demonstrated a cryoprotective effect in spermatozoa involving the Rho/ROCK pathway during freeze-thawing. Furthermore, the addition of 0.5 μg/mL RhoA recombinant protein to the cryoprotective fluid improved rooster semen quality and subcellular structural homeostasis after freeze-thawing via the Rho/ROCK pathway. This pathway may regulate the dynamic reorganization of the actin cytoskeleton by regulating the cofilin phosphorylation.

Cucurbitacins Elicit Anti-Platelet Activity via Perturbation of the Cytoskeleton and Integrin Function.

Cucurbitacins are dietary compounds that have been shown to elicit a range of anti-tumour, anti-inflammatory and anti-atherosclerotic activities. Originally identified as signal transducer and activator of transcription, STAT, inhibitors, a variety of mechanisms of action have since been described, including dysregulation of the actin cytoskeleton and disruption of integrin function. Integrin outside-in signalling and cytoskeletal rearrangements are critical for the propagation of stable thrombus formation and clot retraction following platelet adhesion at the site of vessel damage. The effects of cucurbitacins on platelet function and thrombus formation are unknown. We report for the first time anti-platelet and anti-thrombotic effects of cucurbitacins B, E and I in human platelets. Treatment of platelets with cucurbitacins resulted in attenuation of platelet aggregation, secretion and fibrinogen binding following stimulation by platelet agonists. Cucurbitacins were also found to potently inhibit other integrin- and cytoskeleton-mediated events, including adhesion, spreading and clot retraction. Further investigation of cytoskeletal dynamics found treatment with cucurbitacins altered cofilin phosphorylation, enhanced activation and increased F actin polymerisation and microtubule assembly. Disruption to cytoskeletal dynamics has been previously shown to impair integrin activation, platelet spreading and clot retraction. Anti-platelet properties of cucurbitacins were found to extend to a disruption of stable thrombus formation, with an increase in thrombi instability and de-aggregation under flow. Our research identifies novel, anti-platelet and anti-thrombotic actions of cucurbitacins that appear to be linked to dysregulation of cytoskeletal dynamics and integrin function.

LIM Kinases, Promising but Reluctant Therapeutic Targets: Chemistry and Preclinical Validation In Vivo.

LIM Kinases are important actors in the regulation of cytoskeleton dynamics by controlling microtubule and actin filament turnover. The signaling pathways involving LIM kinases for actin filament remodeling are well established. They are downstream effectors of small G proteins of the Rho-GTPases family and have become promising targets for the treatment of several major diseases because of their position at the lower end of these signaling cascades. Cofilin, which depolymerizes actin filaments, is the best-known substrate of these enzymes. The phosphorylation of cofilin to its inactive form by LIM kinases avoids actin filament depolymerization. The balance between phosphorylated and non-phosphorylated cofilin is thought to play an important role in tumor cell invasion and metastasis. Since 2006, many small molecules have been developed for LIMK inhibition, and in this review article, we will discuss the structure–activity relationships of the few inhibitor families that have been tested in vivo on different pathological models.

Cervical Cancer Cells-Derived Extracellular Vesicles Containing microRNA-146a-5p Affect Actin Dynamics to Promote Cervical Cancer Metastasis by Activating the Hippo-YAP Signaling Pathway via WWC2.

Application of extracellular vesicles (EVs) for cancer treatment has been well-documented. We probed into the potential role of cervical cancer cells-secreted EVs by transferring miR-146a-5p in cervical cancer. After characterization of miR-146a-5p expression in clinical cervical cancer tissue samples, gain- and loss-of-function experiments were implemented to test the effect of miR-146a-5p on the invasion, epithelial-mesenchymal transition (EMT), and anoikis in cervical cancer cells. EVs were isolated from high-metastatic cervical cancer cells, after which their effects on the malignant behaviors of low-metastatic cervical cancer cells were assessed in a co-culture system. Luciferase assay was implemented to validate the putative binding relationship between miR-146a-5p and WWC2, followed by further investigation of downstream pathway (Hippo-YAP). Finally, nude mouse lung metastasis model was developed for in vivo validation. miR-146a-5p was elevated in cervical cancer tissues and high miR-146a-5p expression promoted the metastatic potential of cervical cancer cells through enhancing their invasiveness and anoikis resistance, and inducing EMT. Furthermore, miR-146a-5p carried by EVs secreted by highly metastatic cervical cancer cells could promote the metastasis of low-metastatic cervical cancer cells. Mechanistically, miR-146a-5p targeted WWC2 to activate YAP, by which it inhibited the phosphorylation of cofilin, and promoted the process of cofilin-mediated depolymerization of F-actin to G-actin. In vivo data demonstrated that EVs-carried miR-146a-5p promoted tumor metastasis through the WWC2/YAP axis. Cancer-derived EVs delivered pro-metastatic miR-146a-5p to regulate the actin dynamics in cervical cancer, thereby leading to cancer metastasis. This experiment highlighted an appealing therapeutic modality for cervical cancer.

Autophagy Protects against Eosinophil Cytolysis and Release of DNA.

The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Eosinophils deposit their damaging products in airway tissue, likely by degranulation and cytolysis. We previously showed that priming blood eosinophils with IL3 strongly increased their cytolysis on aggregated IgG. Conversely, IL5 priming did not result in significant eosinophil cytolysis in the same condition. Therefore, to identify critical events protecting eosinophils from cell cytolysis, we examined the differential intracellular events between IL5- and IL3-primed eosinophils interacting with IgG. We showed that both IL3 and IL5 priming increased the eosinophil adhesion to IgG, phosphorylation of p38, and production of reactive oxygen species (ROS), and decreased the phosphorylation of cofilin. However, autophagic flux as measured by the quantification of SQSTM1-p62 and lipidated-MAP1L3CB over time on IgG, with or without bafilomycin-A1, was higher in IL5-primed compared to IL3-primed eosinophils. In addition, treatment with bafilomycin-A1, an inhibitor of granule acidification and autophagolysosome formation, enhanced eosinophil cytolysis and DNA trap formation in IL5-primed eosinophils. Therefore, this study suggests that increased autophagy in eosinophils protects from cytolysis and the release of DNA, and thus limits the discharge of damaging intracellular eosinophilic contents.

Role of MrgprB2 in Rosacea-Like Inflammation in Mice: Modulation by β-Arrestin 2

Cathelicidin LL-37‒mediated activation of mast cells (MCs) has been implicated in the pathogenesis of rosacea, but the receptor involved and the mechanism of its activation and regulation remain unknown. We found that skin biopsies from patients with rosacea display higher frequencies of MCs expressing MRGPRX2 (mouse counterpart MrgprB2) than normal skin. Intradermal injection of LL-37 in wild-type mice resulted in MC recruitment, expression of inflammatory mediators, and development of rosacea-like inflammation. Theseresponses were substantially reduced in MrgprB2‒/‒ mice and abolished in MC deficient Wsh/Wsh mice. β-arrestin 2 is an adaptor protein that regulates G protein-coupled receptor function by receptor desensitization and also by activation of downstream signaling. We found that LL-37‒induced rosacea-like inflammation was significantly reduced in mice with MC-specific deletion of β-arrestin 2 compared with that in control mice. Interestingly, the absence of β-arrestin 2 resulted in enhanced cofilin phosphorylation and substantial inhibition of LL-37‒induced chemotaxis of mouse peritoneal MCs. Furthermore, LL-37‒induced extracellular signal‒regulated kinase 1/2 phosphorylation, NF-κB activation, and proinflammatory cytokine/chemokine production were reduced in β-arrestin 2‒/‒ peritoneal MCs compared with those in wild-type cells. These findings suggest that MRGPRX2/B2 participates in rosacea and that β-arrestin 2 contributes to its pathogenesis by promoting cofilin dephosphorylation, extracellular signal‒regulated kinase 1/2 and NF-κB phosphorylation, MC chemotaxis, and chemokine/cytokine generation.

Overexpression of REST Causes Neuronal Injury and Decreases Cofilin Phosphorylation in Mice.

RE1-silencing transcription factor (REST) is known to silence target genes involved in synaptic plasticity and neuronal differentiation. Although previous studies implicate REST in neurodegenerative diseases, its function in the progression of Alzheimer's disease (AD) is uncertain. The aim of the present work was to explore the mechanisms of AD and determine whether and how REST was involved in the pathogenesis of AD. We investigated the differentially expressed genes and key transcription factors in AD using bioinformatics analysis. In addition, we assessed the expression of REST under the influence of AD-related factors. Mice overexpressing REST were generated and analyzed by proteomics analysis. We used transmission electron microscopy, Golgi-cox staining, immunohistochemistry, and western blotting to examine the impact of REST on neurons. The results of bioinformatics analysis revealed REST as a hub transcriptional regulator in AD. We demonstrate that the mRNA expression of REST was significantly upregulated compared with that in the control groups, not only in AD patients but also in APP/PS1 transgenic mice, lipopolysaccharide-induced neuroinflammatory mice, and oxidative and glutamate stressed neurons. Using proteomics analysis, we showed that the upregulation of REST increased the expression of genes involved in apoptotic and mitochondrial pathways. Long-term overexpression of REST significantly reduced the number of dendritic spines and increased the mitochondrial defect and apoptosis. Reduction of the cofilin phosphorylation may be one of its mechanisms, and cofilin activity could be affected through the P38 MAPK/CREB signaling pathway. These results demonstrated the possible mechanism underlying AD and indicated REST as a potential therapeutic target for AD.