Presynaptic structural and functional plasticity are coupled by convergent Rap1 signaling.

Dynamic presynaptic actin remodeling drives structural and functional plasticity at synapses, but the underlying mechanisms remain largely unknown. Previous work has shown that actin regulation via Rac1 guanine exchange factor (GEF) Vav signaling restrains synaptic growth via bone morphogenetic protein (BMP)-induced receptor macropinocytosis and mediates synaptic potentiation via mobilization of reserve pool vesicles in presynaptic boutons. Here, we find that Gef26/PDZ-GEF and small GTPase Rap1 signaling couples the BMP-induced activation of Abelson kinase to this Vav-mediated macropinocytosis. Moreover, we find that adenylate cyclase Rutabaga (Rut) signaling via exchange protein activated by cAMP (Epac) drives the mobilization of reserve pool vesicles during post-tetanic potentiation (PTP). We discover that Rap1 couples activation of Rut-cAMP-Epac signaling to Vav-mediated synaptic potentiation. These findings indicate that Rap1 acts as an essential, convergent node for Abelson kinase and cAMP signaling to mediate BMP-induced structural plasticity and activity-induced functional plasticity via Vav-dependent regulation of the presynaptic actin cytoskeleton.

Activation of the MEK/ERK Pathway Mediates the Inhibitory Effects of Silvestrol on Nasopharyngeal Carcinoma Cells via RAP1A, HK2, and GADD45A.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor associated with Epstein-Barr virus (EBV) infection. Chemoradiotherapy is the mainstream treatment for locally advanced NPC, and chemotherapeutic drugs are an indispensable part of NPC treatment. However, the toxic side-effects of chemotherapy drugs limit their therapeutic value, and new chemotherapy drugs are urgently needed for NPC. Silvestrol, an emerging natural plant anticancer molecule, has shown promising antitumor activity in breast cancer, melanoma, liver cancer, and other tumor types by promoting apoptosis in cancer cells to a greater extent than in normal cells. However, the effects of silvestrol on NPC and its possible molecular mechanisms have yet to be fully explored. METHODS: Cell counting kit-8 (CCK-8), cell scratch, flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU), and Western blot (WB) assays were used to evaluate the effects of silvestrol on the cell viability, cell cycle, apoptosis, and migration of NPC cells. RNA sequencing (RNA-Seq) was used to study the effect of extracellular signal-regulated kinase (ERK) inhibitors on the cell transcriptome, and immunohistochemistry (IHC) to assess protein expression levels in patient specimens. RESULTS: Silvestrol inhibited cell migration and DNA replication of NPC cells, while promoting the expression of cleaved caspase-3, apoptosis, and cell cycle arrest. Furthermore, silvestrol altered the level of ERK phosphorylation. The ERK-targeted inhibitor LY3214996 attenuated silvestrol-mediated inhibition of NPC cell proliferation but not migration. Analysis of RNA-Seq data and WB were used to identify and validate the downstream regulatory targets of silvestrol. Expression of GADD45A, RAP1A, and hexokinase-II (HK2) proteins was inhibited by silvestrol and LY3214996. Finally, IHC revealed that GADD45A, RAP1A, and HK2 protein expression was more abundant in cancer tissues than in non-tumor tissues. CONCLUSIONS: Silvestrol inhibits the proliferation of NPC cells by targeting ERK phosphorylation. However, the inhibition of NPC cell migration by silvestrol was independent of the Raf-MEK-ERK pathway. RAP1A, HK2, and GADD45A may be potential targets for the action of silvestrol.

EspP2 Regulates the Adhesion of Glaesserella parasuis via Rap1 Signaling Pathway.

Different levels of EspP2 expression are seen in strains of Glaesserella parasuis with high and low pathogenicity. As a potential virulence factor for G. parasuis, the pathogenic mechanism of EspP2 in infection of host cells is not clear. To begin to elucidate the effect of EspP2 on virulence, we used G. parasuis SC1401 in its wild-type form and SC1401, which was made EspP2-deficient. We demonstrated that EspP2 causes up-regulation of claudin-1 and occludin expression, thereby promoting the adhesion of G. parasuis to host cells; EspP2-deficiency resulted in significantly reduced adhesion of G. parasuis to cells. Transcriptome sequencing analysis of EspP2-treated PK15 cells revealed that the Rap1 signaling pathway is stimulated by EspP2. Blocking this pathway diminished occludin expression and adhesion. These results indicated that EspP2 regulates the adhesion of Glaesserella parasuis via Rap1 signaling pathway.

Spatial Mechano-Signaling Regulation of GTPases through Non-Degradative Ubiquitination.

Blood flow produces shear stress exerted on the endothelial layer of the vessels. Spatial characterization of the endothelial proteome is required to uncover the mechanisms of endothelial activation by shear stress, as blood flow varies in the vasculature. An integrative ubiquitinome and proteome analysis of shear-stressed endothelial cells demonstrated that the non-degradative ubiquitination of several GTPases is regulated by mechano-signaling. Spatial analysis reveals increased ubiquitination of the small GTPase RAP1 in the descending aorta, a region exposed to laminar shear stress. The ubiquitin ligase WWP2 is identified as a novel regulator of RAP1 ubiquitination during shear stress response. Non-degradative ubiquitination fine-tunes the function of GTPases by modifying their interacting network. Specifically, WWP2-mediated RAP1 ubiquitination at lysine 31 switches the balance from the RAP1/ Talin 1 (TLN1) toward RAP1/ Afadin (AFDN) or RAP1/ RAS Interacting Protein 1 (RASIP1) complex formation, which is essential to suppress shear stress-induced reactive oxygen species (ROS) production and maintain endothelial barrier integrity. Increased ROS production in endothelial cells in the descending aorta of endothelial-specific Wwp2-knockout mice leads to increased levels of oxidized lipids and inflammation. These results highlight the importance of the spatially regulated non-degradative ubiquitination of GTPases in endothelial mechano-activation.

Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.

Atorvastatin-mediated inhibition of prenylation of Rab27b and Rap1a in platelets attenuates their prothrombotic capacity and modulates clot structure.

Statins inhibit the mevalonate pathway by impairing protein prenylation via depletion of lipid geranylgeranyl diphosphate (GGPP). Rab27b and Rap1a are small GTPase proteins involved in dense granule secretion, platelet activation, and regulation. We analyzed the impact of statins on prenylation of Rab27b and Rap1a in platelets and the downstream effects on fibrin clot properties. Whole blood thromboelastography revealed that atorvastatin (ATV) delayed clot formation time (P < .005) and attenuated clot firmness (P < .005). ATV pre-treatment inhibited platelet aggregation and clot retraction. Binding of fibrinogen and P-selectin exposure on stimulated platelets was significantly lower following pre-treatment with ATV (P < .05). Confocal microscopy revealed that ATV significantly altered the structure of platelet-rich plasma clots, consistent with the reduced fibrinogen binding. ATV enhanced lysis of Chandler model thrombi 1.4-fold versus control (P < .05). Western blotting revealed that ATV induced a dose-dependent accumulation of unprenylated Rab27b and Rap1a in the platelet membrane. ATV dose-dependently inhibited ADP release from activated platelets. Exogenous GGPP rescued the prenylation of Rab27b and Rap1a, and partially restored the ADP release defect, suggesting these changes arise from reduced prenylation of Rab27b. These data demonstrate that statins attenuate platelet aggregation, degranulation, and binding of fibrinogen thereby having a significant impact on clot contraction and structure.

What is the context? Statins such as Atorvastatin (ATV) are 3-hydroxy, 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which block the cholesterol biosynthetic pathway to lower total serum levels and LDL-cholesterol.The cholesterol pathway also provides a supply of isoprenoids (farnesyl and geranylgeranyl) for the prenylation of signaling molecules, which include the families of Ras and Rho small GTPases.Prenyl groups provide a membrane anchor that is essential for the correct membrane localization and function of these proteins.Statins deplete cells of lipid geranylgeranyl diphosphate (GGPP) thereby inhibiting progression of the mevalonate pathway and prenylation of proteins.Rab27b and Rap1 are small GTPase proteins in platelets that are involved in the secretion of platelet granules and integrin activation.What is new?In this study, we found that ATV impairs prenylation of Rab27b and Rap1a and attenuates platelet function.These effects were partially rescued by GGPP, indicating the involvement of the mevalonate pathway.Platelet aggregation and degranulation was significantly attenuated by ATV.The impact of statins on platelet function altered clot formation, structure and contraction generating a clot that was more susceptible to degradation.What is the impact?This study demonstrates a novel mechanism whereby statins alter platelet responses and ultimately clot structure and stability.

Homology directed telomere clustering, ultrabright telomere formation and nuclear envelope rupture in cells lacking TRF2B and RAP1.

Double-strand breaks (DSBs) due to genotoxic stress represent potential threats to genome stability. Dysfunctional telomeres are recognized as DSBs and are repaired by distinct DNA repair mechanisms. RAP1 and TRF2 are telomere binding proteins essential to protect telomeres from engaging in homology directed repair (HDR), but how this occurs remains unclear. In this study, we examined how the basic domain of TRF2 (TRF2B) and RAP1 cooperate to repress HDR at telomeres. Telomeres lacking TRF2B and RAP1 cluster into structures termed ultrabright telomeres (UTs). HDR factors localize to UTs, and UT formation is abolished by RNaseH1, DDX21 and ADAR1p110, suggesting that they contain DNA-RNA hybrids. Interaction between the BRCT domain of RAP1 and KU70/KU80 is also required to repress UT formation. Expressing TRF2∆B in Rap1-/- cells resulted in aberrant lamin A localization in the nuclear envelope and dramatically increased UT formation. Expressing lamin A phosphomimetic mutants induced nuclear envelope rupturing and aberrant HDR-mediated UT formation. Our results highlight the importance of shelterin and proteins in the nuclear envelope in repressing aberrant telomere-telomere recombination to maintain telomere homeostasis.

Rap1A accelerates homocysteine-induced ANA-1 cells inflammation via synergy of FoxO1 and DNMT3a.

Abnormal elevation of homocysteine (Hcy) level accelerates atherosclerosis through promote macrophage inflammation, while the precise mechanisms remain to be well elucidated. Previous study revealed that Rap1A is involved in the development of atherosclerosis, but little is known regarding the regulation of macrophage inflammation induced by Hcy and its potential mechanisms. In the present study, we demonstrated that Hcy upregulates Rap1A expression and knockdown of Rap1A inhibited pro-inflammatory cytokines IL-6 and TNF-α levels in ANA-1 cells. Mechanistically, DNMT3a-mediated DNA hypomethylation of Rap1A promoter accelerates Hcy-induced ANA-1 cells inflammation. Furthermore, FoxO1 transcriptionally activate Rap1A by direct binding to its promoter. More importantly, Hcy could enhance FoxO1 interaction with DNMT3a and synergistically promote the expression of Rap1A resulting in accelerate ANA-1 cells inflammation. These data indicate that Rap1A is a novel and important regulator in Hcy-induced ANA-1 cells inflammation.

CAP1 (cyclase-associated protein 1) mediates the cyclic AMP signals that activate Rap1 in stimulating matrix adhesion of colon cancer cells.

We previously reported that CAP1 (Cyclase-Associated Protein 1) regulates matrix adhesion in mammalian cells through FAK (Focal Adhesion Kinase). More recently, we discovered a phosphor-regulation mechanism for CAP1 through the Ser307/Ser309 tandem site that is of critical importance for all CAP1 functions. However, molecular mechanisms underlying the CAP1 function in adhesion and its regulation remain largely unknown. Here we report that Rap1 also facilitates the CAP1 function in adhesion, and more importantly, we identify a novel signaling pathway where CAP1 mediates the cAMP signals, through the cAMP effectors Epac (Exchange proteins directly activated by cAMP) and PKA (Protein Kinase A), to activate Rap1 in stimulating matrix adhesion in colon cancer cells. Knockdown of CAP1 led to opposite adhesion phenotypes in SW480 and HCT116 colon cancer cells, with reduced matrix adhesion and reduced FAK and Rap1 activities in SW480 cells while it stimulated matrix adhesion as well as FAK and Rap1 activities in HCT116 cells. Importantly, depletion of CAP1 abolished the stimulatory effects of the cAMP activators forskolin and isoproterenol, as well as that of Epac and PKA, on matrix adhesion in both cell types. Our results consistently support a required role for CAP1 in the cAMP activation of Rap1. Identification of the key role for CAP1 in linking the major second messenger cAMP to activation of Rap1 in stimulating adhesion, which may potentially also regulate proliferation in other cell types, not only vertically extends our knowledge on CAP biology, but also carries important translational potential for targeting CAP1 in cancer therapeutics.

Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia.

Excessive hepatic glucose production contributes to the development of hyperglycemia and is a key feature of type 2 diabetes. Here, we report that activation of hepatocyte Rap1a suppresses gluconeogenic gene expression and glucose production, whereas Rap1a silencing stimulates them. Rap1a activation is suppressed in obese mouse liver, and restoring its activity improves glucose intolerance. As Rap1a's membrane localization and activation depends on its geranylgeranylation, which is inhibited by statins, we show that statin-treated hepatocytes and the human liver have lower active-Rap1a levels. Similar to Rap1a inhibition, statins stimulate hepatic gluconeogenesis and increase fasting blood glucose in obese mice. Geranylgeraniol treatment, which acts as the precursor for geranylgeranyl isoprenoids, restores Rap1a activity and improves statin-mediated glucose intolerance. Mechanistically, Rap1a activation induces actin polymerization, which suppresses gluconeogenesis by Akt-mediated FoxO1 inhibition. Thus, Rap1a regulates hepatic glucose homeostasis, and blocking its activity, via lowering geranylgeranyl isoprenoids, contributes to statin-induced glucose intolerance.

Irisin enhances chondrogenic differentiation of human mesenchymal stem cells via Rap1/PI3K/AKT axis.

BACKGROUND: Human mesenchymal stem cells (hMSCs) have been proven to have inherent chondrogenic differentiation potential, which appears to be used in cartilage regeneration. Increasing evidence suggests that irisin enhances osteoblast differentiation of MSCs, but little is known about its potential on chondrogenic differentiation. METHODS: In the study, we investigated the effects of irisin on chondrogenic differentiation of hMSCs using a high-density pellet culture system. The cartilage pellets were evaluated by morphology, and the metabolism of cartilage matrix was detected by qPCR, western blot and immunohistochemistry. Next, RNA-seq was performed to explore the underlying mechanism. Furthermore, using the transduction of plasmid, miRNAs mimics and inhibitor, the activation of Rap1/PI3K/AKT axis, the expression level of SIPA1L2, and the functional verification of miR-125b-5p were detected on day 7 of chondrogenic differentiation of hMSCs. RESULTS: Compared with the controls, we found that irisin treatment could significantly enhance the chondrogenic differentiation of hMSCs, enlarge the induced-cartilage tissue and up-regulate the expression levels of cartilage markers. RNA-seq indicated that irisin activated the Rap1 and PI3K/AKT signaling pathway, and the lower expression level of SIPA1L2 and the higher expression level of miR-125b-5p were found in irisin-treated group. Further, we found that irisin treatment could up-regulate the expression level of miR-125b-5p, targeting SIPA1L2 and consequently activating the Rap1/PI3K/AKT axis on the process of chondrogenic differentiation of hMSCs. CONCLUSIONS: Collectively, our study reveals that irisin can enhance chondrogenic differentiation of hMSCs via the Rap1/PI3K/AKT pathway, suggesting that irisin possesses prospects in cartilage regeneration.

MiR-1224-5p modulates osteogenesis by coordinating osteoblast/osteoclast differentiation via the Rap1 signaling target ADCY2.

MicroRNAs (miRNAs) broadly regulate normal biological functions of bone and the progression of fracture healing and osteoporosis. Recently, it has been reported that miR-1224-5p in fracture plasma is a potential therapy for osteogenesis. To investigate the roles of miR-1224-5p and the Rap1 signaling pathway in fracture healing and osteoporosis development and progression, we used BMMs, BMSCs, and skull osteoblast precursor cells for in vitro osteogenesis and osteoclastogenesis studies. Osteoblastogenesis and osteoclastogenesis were detected by ALP, ARS, and TRAP staining and bone slice resorption pit assays. The miR-1224-5p target gene was assessed by siRNA-mediated target gene knockdown and luciferase reporter assays. To explore the Rap1 pathway, we performed high-throughput sequencing, western blotting, RT-PCR, chromatin immunoprecipitation assays and immunohistochemical staining. In vivo, bone healing was judged by the cortical femoral defect, cranial bone defect and femoral fracture models. Progression of osteoporosis was evaluated by an ovariectomy model and an aged osteoporosis model. We discovered that the expression of miR-1224-5p was positively correlated with fracture healing progression. Moreover, in vitro, overexpression of miR-1224-5p slowed Rankl-induced osteoclast differentiation and promoted osteoblast differentiation via the Rap1-signaling pathway by targeting ADCY2. In addition, in vivo overexpression of miR-1224-5p significantly promoted fracture healing and ameliorated the progression of osteoporosis caused by estrogen deficiency or aging. Furthermore, knockdown of miRNA-1224-5p inhibited bone regeneration in mice and accelerated the progression of osteoporosis in elderly mice. Taken together, these results identify miR-1224-5p as a key bone osteogenic regulator, which may be a potential therapeutic target for osteoporosis and fracture nonunion.

Phostensin enables lymphocyte integrin activation and population of peripheral lymphoid organs.

Rap1 GTPase drives assembly of the Mig-10/RIAM/Lamellipodin (MRL protein)-integrin-talin (MIT) complex that enables integrin-dependent lymphocyte functions. Here we used tandem affinity tag-based proteomics to isolate and analyze the MIT complex and reveal that Phostensin (Ptsn), a regulatory subunit of protein phosphatase 1, is a component of the complex. Ptsn mediates dephosphorylation of Rap1, thereby preserving the activity and membrane localization of Rap1 to stabilize the MIT complex. CRISPR/Cas9-induced deletion of PPP1R18, which encodes Ptsn, markedly suppresses integrin activation in Jurkat human T cells. We generated apparently healthy Ppp1r18-/- mice that manifest lymphocytosis and reduced population of peripheral lymphoid tissues ascribable, in part, to defective activation of integrins αLß2 and α4ß7. Ppp1r18-/- T cells exhibit reduced capacity to induce colitis in a murine adoptive transfer model. Thus, Ptsn enables lymphocyte integrin-mediated functions by dephosphorylating Rap1 to stabilize the MIT complex. As a consequence, loss of Ptsn ameliorates T cell-mediated colitis.

Rap1a Activity Elevated the Impact of Endogenous AGEs in Diabetic Collagen to Stimulate Increased Myofibroblast Transition and Oxidative Stress.

Diabetics have an increased risk for heart failure due to cardiac fibroblast functional changes occurring as a result of AGE/RAGE signaling. Advanced glycation end products (AGEs) levels are higher in diabetics and stimulate elevated RAGE (receptor for AGE) signaling. AGE/RAGE signaling can alter the expression of proteins linked to extracellular matrix (ECM) remodeling and oxidative stressors. Our lab has identified a small GTPase, Rap1a, that may overlap the AGE/RAGE signaling pathway. We sought to determine the role Rap1a plays in mediating AGE/RAGE changes and to assess the impact of isolated collagen on further altering these changes. Primary cardiac fibroblasts from non-diabetic and diabetic mice with and without RAGE expression and from mice lacking Rap1a were cultured on tail collagen extracted from non-diabetic or diabetic mice, and in addition, cells were treated with Rap1a activator, EPAC. Protein analyses were performed for changes in RAGE-associated signaling proteins (RAGE, PKC-ζ, ERK1/2) and downstream RAGE signaling outcomes (α-SMA, NF-κB, SOD-2). Increased levels of endogenous AGEs within the diabetic collagen and increased Rap1a activity promoted myofibroblast transition and oxidative stress, suggesting Rap1a activity elevated the impact of AGEs in the diabetic ECM to stimulate myofibroblast transition and oxidative stress.

Rap1A, Rap1B, and ß-Adrenergic Signaling in Autologous HCT: A Randomized Controlled Trial of Propranolol.

Successful hematopoietic cell transplantation (HCT) depends on rapid engraftment of the progenitor and stem cells that will reestablish hematopoiesis. Rap1A and Rap1B are two closely related small GTPases that may affect platelet and neutrophil engraftment during HCT through their roles in cell adhesion and migration. ß-adrenergic signaling may regulate the participation of Rap1A and Rap1B in engraftment through their inhibition or activation. We conducted a correlative study of a randomized controlled trial evaluating the effects of the nonselective ß-antagonist propranolol on expression and prenylation of Rap1A and Rap1B during neutrophil and platelet engraftment in 25 individuals receiving an autologous HCT for multiple myeloma. Propranolol was administered for 1 week prior to and 4 weeks following HCT. Blood was collected 7 days (baseline) and 2 days (Day -2) before HCT, and 28 days after HCT (Day +28). Circulating polymorphonuclear cells (PMNC) were isolated and analyzed via immunoblotting to determine levels of prenylated and total Rap1A versus Rap1B. Twelve participants were randomized to the intervention and 13 to the control. Rap1A expression significantly correlated with Rap1B expression. Rap1B expression significantly correlated with slower platelet engraftment; however, this association was not observed in the propranolol-treated group. There were no significant associations between neutrophil engraftment and Rap1A or Rap1B expression. Post hoc exploratory analyses did not reveal an association between social health variables and Rap1A or Rap1B expression. This study identifies a greater regulatory role for Rap1B than Rap1A in platelet engraftment and suggests a possible role for ß-adrenergic signaling in modulating Rap1B function during HCT.

Rap1 prevents colitogenic Th17 cell expansion and facilitates Treg cell differentiation and distal TCR signaling.

T-cell-specific Rap1 deletion causes spontaneous colitis in mice. In the present study, we revealed that Rap1 deficiency in T cells impaired the preceding induction of intestinal RORγt+ Treg cells. In the large intestinal lamina propria (LILP) of T-cell-specific Rap1-knockout mice (Rap1KO mice), Th17 cells were found to increase in a microbiota-dependent manner, and the inhibition of IL-17A production prevented the development of colitis. In the LILP of Rap1KO mice, RORγt+ Treg cells were scarcely induced by 4 weeks of age. The expression of CTLA-4 on Rap1-deficient Treg cells was reduced and the expression of CD80 and CD86 on dendritic cells was consequently elevated in Rap1KO mice. When cultured under each polarizing condition, Rap1-deficient naïve CD4+ T cells did not show biased differentiation into Th17 cells; their differentiation into Treg cells as well as Th1 and Th2 cells was lesser than that of wild-type cells. Rap1-deficient naïve CD4+ T cells were found to exhibit the defective nuclear translocation of NFAT and formation of actin foci in response to TCR engagement. These data suggest that Rap1 amplifies the TCR signaling required for Treg-mediated control of intestinal colitogenic Th17 responses.

ArhGEF12 activates Rap1A and not RhoA in human dermal microvascular endothelial cells to reduce tumor necrosis factor-induced leak.

Overwhelming inflammation in the setting of acute critical illness induces capillary leak resulting in hypovolemia, edema, tissue dysoxia, organ failure and even death. The tight junction (TJ)-dependent capillary barrier is regulated by small GTPases, but the specific regulatory molecules most active in this vascular segment under such circumstances are not well described. We set out to identify GTPase regulatory molecules specific to endothelial cells (EC) that form TJs. Transcriptional profiling of confluent monolayers of TJ-forming human dermal microvascular ECs (HDMECs) and adherens junction only forming-human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is basally expressed at higher levels and is only downregulated in HDMECs by junction-disrupting tumor necrosis factor (TNF). HDMECs depleted of ArhGEF12 by siRNA demonstrate a significantly exacerbated TNF-induced decrease in trans-endothelial electrical resistance and disruption of TJ continuous staining. ArhGEF12 is established as a RhoA-GEF in HUVECs and its knock down would be expected to reduce RhoA activity and barrier disruption. Pulldown of active GEFs from HDMECs depleted of ArhGEF12 and treated with TNF show decreased GTP-bound Rap1A after four hours but increased GTP-bound RhoA after 12 h. In cell-free assays, ArhGEF12 immunoprecipitated from HDMECs is able to activate both Rap1A and RhoA, but not act on Rap2A-C, RhoB-C, or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in TJ-forming HDMECs, ArhGEF12 selectively activates Rap1A to limit capillary barrier disruption in a mechanism independent of cAMP-mediated Epac1 activation.

Direct Binding of Rap1 to Talin1 and to MRL Proteins Promotes Integrin Activation in CD4+ T Cells.

Agonist-induced Rap1 GTP loading results in integrin activation involved in T cell trafficking and functions. MRL proteins Rap1-interacting adapter molecule (RIAM) and lamellipodin (LPD) are Rap1 effectors that can recruit talin1 to integrins, resulting in integrin activation. Recent work also implicates direct Rap1-talin1 interaction in integrin activation. Here, we analyze in mice the connections between Rap1 and talin1 that support integrin activation in conventional CD4+ T (Tconv) and CD25HiFoxp3+CD4+ regulatory T (Treg) cells. Talin1(R35E, R118E) mutation that disrupts both Rap1 binding sites results in a partial defect in αLß2, α4ß1, and α4ß7 integrin activation in both Tconv and Treg cells with resulting defects in T cell homing. Talin1(R35E,R118E) Tconv manifested reduced capacity to induce colitis in an adoptive transfer mouse model. Loss of RIAM exacerbates the defects in Treg cell function caused by the talin1(R35E,R118E) mutation, and deleting both MRL proteins in combination with talin1(R35E,R118E) phenocopy the complete lack of integrin activation observed in Rap1a/b-null Treg cells. In sum, these data reveal the functionally significant connections between Rap1 and talin1 that enable αLß2, α4ß1, and α4ß7 integrin activation in CD4+ T cells.

Elevated RAP1A expression correlates with the severity of acute GVHD after umbilical cord blood transplantation.

BACKGROUND: Acute graft-versus-host disease (aGVHD) is a complication of allogeneic hematopoietic stem cell transplantation. Ras-related protein 1A (RAP1A) has been recently identified as a novel oncoprotein in several human malignancies. However, its specific role in aGVHD remains unclear. OBJECTIVE: To study the role of RAP1A in the pathogenesis of aGVHD. MATERIAL AND METHODS: Study participants included six patients with grade 2-4 aGVHD, 13 patients with grade 1 aGVHD, 11 patients without aGVHD, and 12 healthy people. The expression level of RAP1A in whole cells was detected by western blot and qRT-PCR. The proportions of CD4+CD25+FoxP3+ Treg cells (T regulatory cells) and the expression of RAP1A in Treg cells in peripheral blood mononuclear cells (PBMCs) were detected by flow cytometry and the levels of related cytokines in the serum was detected by cytometric bead array. RESULTS: We found the level of RAP1A was higher in patients than in healthy individuals. A negative correlation was noted between RAP1A and the number of Treg cells. In addition, the level of IL-10 in patients with grade 2-4 aGVHD was significantly lower than that in healthy donors, however, the level of TNF-ɑ in patients with grade 2-4 aGVHD was higher. Furthermore, we found a negative correlation between levels of IL-10 and RAP1A, and a positive correlation between TNF-ɑ and RAP1A. CONCLUSION: The expression of RAP1A in patients with aGVHD was significantly increased, and shows potential as a target for the prevention and treatment of aGVHD.

FABP4 activates the JAK2/STAT2 pathway via Rap1a in the homocysteine-induced macrophage inflammatory response in ApoE-/- mice atherosclerosis.

Atherosclerosis is a chronic inflammatory vascular disease, and inflammation plays a critical role in its formation and progression. Elevated serum homocysteine (Hcy) is an independent risk factor for atherosclerosis. Previous studies have shown that fatty acid binding protein 4 (FABP4) plays an important role in macrophage inflammation and lipid metabolism in atherosclerosis induced by Hcy. However, the underlying molecular mechanism of FABP4 in Hcy-induced macrophage inflammation remains unknown. In this study, we found that FABP4 activated the Janus kinase 2/signal transducer and activator of transcription 2 (JAK2/STAT2) pathway in macrophage inflammation induced by Hcy. Of note, we further observed that ras-related protein Rap-1a (Rap1a) induced the Tyr416 phosphorylation and membrane translocation of non-receptor tyrosine kinase (c-Src) to activate the JAK2/STAT2 pathway. In addition, the suppressor of cytokine signaling 1 (SOCS1)-a transcriptional target of signal transducer and activator of transcription (STATs) inhibited the JAK2/STAT2 pathway and Rap1a expression via a negative feedback loop. In summary, these results demonstrated that FABP4 promotes c-Src phosphorylation and membrane translocation via Rap1a to activate the JAK2/STAT2 pathway, contributing to Hcy-accelerated macrophage inflammation in ApoE-/- mice.