Rac1 Signaling Research Articles

Loss-of-Function Mutations in ELMO2 Cause Intraosseous Vascular Malformation by Impeding RAC1 Signaling

Vascular malformations are non-neoplastic expansions of blood vessels that arise due to errors during angiogenesis. They are a heterogeneous group of sporadic or inherited vascular disorders characterized by localized lesions of arteriovenous, capillary, or lymphatic origin. Vascular malformations that occur inside bone tissue are rare. Herein, we report loss-of-function mutations in ELMO2 (which translates extracellular signals into cellular movements) that are causative for autosomal-recessive intraosseous vascular malformation (VMOS) in five different families. Individuals with VMOS suffer from life-threatening progressive expansion of the jaw, craniofacial, and other intramembranous bones caused by malformed blood vessels that lack a mature vascular smooth muscle layer. Analysis of primary fibroblasts from an affected individual showed that absence of ELMO2 correlated with a significant downregulation of binding partner DOCK1, resulting in deficient RAC1-dependent cell migration. Unexpectedly, elmo2-knockout zebrafish appeared phenotypically normal, suggesting that there might be human-specific ELMO2 requirements in bone vasculature homeostasis or genetic compensation by related genes. Comparative phylogenetic analysis indicated that elmo2 originated upon the appearance of intramembranous bones and the jaw in ancestral vertebrates, implying that elmo2 might have been involved in the evolution of these novel traits. The present findings highlight the necessity of ELMO2 for maintaining vascular integrity, specifically in intramembranous bones.

Abstract 1669: Inhibition of the Rac1/PAK pathway sensitizes pancreatic cancer cells to gamma-irradiation

Abstract Radiation therapy is a staple treatment for pancreatic cancers. However, owing to the intrinsic radioresistance of pancreatic cancer cells, radiation therapy often fails to increase survival of pancreatic cancer patients. Radiation impedes cancer cells by inducing DNA damage, which can activate cell cycle checkpoints. Normal cells possess both G1/S and G2/M checkpoints. However, cancer cells are often defective in G1/S checkpoint due to mutations/alterations in the key regulators of this checkpoint. Accordingly, our results show that normal pancreatic ductal cells respond to ionizing radiation (IR) with activation of both checkpoints whereas pancreatic cancer cells respond to IR with G2/M arrest only. Overexpression/hyperactivation of Rac1 GTPase is detected in the majority of pancreatic cancers. Rac1 plays important roles in survival and Ras mediated transformation. Here, we show that Rac1 and its downstream effector, the PAK kinases, also plays a critical role in the response of pancreatic cancer cells to IR. Inhibition of Rac1/PAK pathway with pharmacological inhibitors (Ehop-016, AZA1, NSC23766, and FRAX597) or a dominant negative mutant of Rac1 (Rac1T17N) blocks activation of the ATM/Chk2 and ATR/Chk1 pathways, abrogates activation of the IR-induced G2/M checkpoint, delays the repair of IR-induced double-stranded DNA breaks, and increases the radiosensitivity of pancreatic cancer cells through induction of apoptosis. These results implicate Rac1 signaling in the survival of pancreatic cancer cells following IR, raising the possibility that this pathway contributes to the intrinsic radioresistance of pancreatic cancer. Citation Format: Michel M. Ouellette, Surinder K. Batra, Ying Yan. Inhibition of the Rac1/PAK pathway sensitizes pancreatic cancer cells to gamma-irradiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1669.

Abstract 31: Metabolic reprogramming in ovarian cancer

Abstract Ovarian cancer is currently the most lethal gynecologic malignancy, with no new significant changes in treatment options for these patients in the last 30 years. Importantly, ovarian cancer patients have increased levels of lysophosphatidic acid (LPA), a bioactive phospholipid in ascites and serum that has been linked to driving oncogenesis and progression of ovarian cancer. Accumulating evidence has implicated Hypoxia-inducible factor-1α (HIF-1α) as a critical mediator of the glycolytic shift observed in cancer cells. However, the precise biological role of LPA in regulating HIF-1α-mediated glycolytic shift is largely unknown. Therefore, we were interested in examining the potential role of LPA in promoting metabolic reprogramming in ovarian cancer via HIF-1α signaling. In this study, we identified a novel mechanism by which LPA upregulates HIF-1α expression via Gαi2 (the gip2 oncogene) in human ovarian cancer cells. This study demonstrates that LPA induces a glycolytic shift via LPA-mediated activation of Gαi2, which causes a subsequent upregulation of HIF-1α in ovarian cancer cells. Additionally, we show that LPA-signaling via Gαi2 induces an increase in the expression of Hexokinase-2 (HK2) and Glucose transporter 1 (GLUT1), which are known targets of HIF-1α. We also demonstrate that LPA induces an increase in extracellular acidification rate (ECAR) in a dose dependent manner in both ovarian cancer cell lines and in patient-derived cells taken from the ascites fluid of ovarian cancer patients using an XFe96 analyzer. Furthermore, we found that inhibition of Rac signaling caused a reduction in LPA-induced ECAR, identifying Rac as a critical downstream component of Gαi2-medidated increase of ECAR. Moreover, using NAC, an inhibitor of redox signaling, we found that this caused a decrease in LPA-induced ECAR in SKOV3-ip cells, indicating that inhibition of redox signaling abolishes LPA-induced ECAR in ovarian cancer cells. Similarly, the use of EUK-134, a scavenger of superoxide and H2O2, also decreased LPA-induced increase in ECAR. Altogether, these results indicate that LPA regulates glycolysis through redox signaling via a Gαi2-Rac-HIF1α-dependent signaling pathway. Most importantly, the Gαi2-Rac-dependent pathway identified in this study more than likely serves as a potential driver of HIF-1α-mediated metabolic changes in ovarian cancer cells and represents a potential target for therapy in these patients. Citation Format: JI HEE HA, Rangasudhagar Radhakrishnan, Jeremy D. Ward, Muralidharan Jayaraman, Danny N. Dhanasekaran. Metabolic reprogramming in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 31.

Methylphenidate-triggered ROS generation promotes caveolae-mediated transcytosis via Rac1 signaling and c-Src-dependent caveolin-1 phosphorylation in human brain endothelial cells.

Methylphenidate (MPH) is an amphetamine-like stimulant commonly prescribed for attention deficit hyperactivity disorder. Despite its widespread use, the cellular/molecular effects of MPH remain elusive. Here, we report a novel direct role of MPH on the regulation of macromolecular flux through human brain endothelial cells (ECs). MPH significantly increased caveolae-mediated transcytosis of horseradish peroxidase through ECs without affecting paracellular permeability. Using FRET-based live cell imaging, together with pharmacological inhibitors and lentiviral-mediated shRNA knockdown, we demonstrate that MPH promoted ROS generation via activation of Rac1-dependent NADPH oxidase (NOX) and c-Src activation at the plasma membrane. c-Src in turn was shown to mediate the phosphorylation of caveolin-1 (Cav1) on Tyr14 leading to enhanced caveolae formation and transendothelial transport. Accordingly, the inhibition of Cav1 phosphorylation by overexpression of a phosphodefective Cav1Y14F mutant or knocking down Cav1 expression abrogated MPH-induced transcytosis. In addition, both vitamin C and inhibition of NOX blocked MPH-triggered vesicular transport. This study, therefore, identifies Rac1/NOX/c-Src-dependent signaling in MPH-induced increase in transendothelial permeability of brain endothelial cell monolayers via caveolae-mediated transcytosis.

Therapeutic effects of the Rho GTPase modulator CNF1 in a model of Parkinson’s disease

Recent evidence suggests an early involvement of dopaminergic (DA) processes and terminals in Parkinson’s disease (PD). The arborization of neurons depends on the actin cytoskeleton, which in turn is regulated by small GTPases of the Rho family, encompassing Rho, Rac and Cdc42 subfamilies. Indeed, some reports point to a role for Rac and Cdc42 signaling in the pathophysiology of inherited parkinsonisms. We thus investigated the potential therapeutic effect of the modulation of cerebral Rho GTPases in PD. Cytotoxic necrotizing factor 1 (CNF1), a 114 kDa protein toxin produced by Escherichia coli, permanently activates RhoA, Rac1 and Cdc42 in intact cells. We report that the modulation of Rho GTPases by CNF1 results in hypertrophy of DA cell processes of cultured substantia nigra neurons, including increase in length, branching and varicosity. In vivo, the treatment corrects long-standing motor and biochemical asymmetries and restores degenerated nigrostriatal DA tissue after 6-hydroxydopamine lesion. We conclude that the pharmacological modulation of Rho GTPases shows neurorestorative potential and represents a promising avenue in the treatment PD. The study also suggests that naturally occurring molecules acting on Rho GTPase signaling, such as some bacterial protein toxins, might play a role in the development of PD.

RAC1 GTPase promotes the survival of breast cancer cells in response to hyper-fractionated radiation treatment.

Radiation therapy is a staple approach for cancer treatment, whereas radioresistance of cancer cells remains a substantial clinical problem. In response to ionizing radiation (IR) induced DNA-damage, cancer cells can sustain/activate pro-survival signaling pathways, leading to apoptotic resistance and induction of cell cycle checkpoint/DNA repair. Previous studies show that Rac1 GTPase is overexpressed/hyperactivated in breast cancer cells and is associated with poor prognosis. Studies from our laboratory reveal that Rac1 activity is necessary for G2/M checkpoint activation and cell survival in response to IR exposure of breast and pancreatic cancer cells. In the present study, we investigated the effect of Rac1 on the survival of breast cancer cells treated with hyper-fractionated radiation (HFR), which is used clinically for cancer treatment. Results in this report indicate that Rac1 protein expression is increased in the breast cancer cells that survived HFR compared to parental cells. Furthermore, this increase of Rac1 is associated with enhanced activities of ERK1/2 and NF-κB signaling pathways and increased levels of anti-apoptotic protein Bcl-xL and Mcl-1, which are downstream targets of ERK1/2 and NF-κB signaling pathways. Using Rac1 specific inhibitor and dominant negative mutant N17Rac1, here we demonstrate that Rac1 inhibition decreases the phosphorylation of ERK1/2 and IκBα, as well as the levels of Bcl-xL and Mcl-1 protein in the HFR-selected breast cancer cells. Moreover, inhibition of Rac1 using either small molecule inhibitor or dominant negative N17Rac1 abrogates clonogenic survival of HFR-selected breast cancer cells and decreases the level of intact PARP, which is indicative of apoptosis induction. Collectively, results in this report suggest that Rac1 signaling is essential for the survival of breast cancer cells subjected to HFR and implicate Rac1 in radioresistance of breast cancer cells. These studies also provide the basis to explore Rac1 as a therapeutic target for radioresistant breast cancer cells.

Ox-LDL induces endothelial dysfunction by promoting Arp2/3 complex expression

Oxidized low-density lipoproteins (ox-LDL) play a critical role in endothelial injury including cytoskeleton reorganization, which is closely related to actin-related protein 2/3 (Arp2/3) complex. The aim of this study was to investigate the role of Arp2/3 complex in ox-LDL-induced endothelial dysfunction. In this study, we found that Arp2 and Arp3 expression was increased under atherosclerotic conditions both in ApoE−/− mice and in ox-LDL-stimulated human coronary artery endothelial cells (HCAECs). Arp2/3 complex inhibitor CK666 significantly reduced ox-LDL-induced ROS generation and cytoskeleton reorganization, and increased NO release in HCAECs. Pretreatment with LOX-1- but not CD36-blocking antibody markedly decreased ox-LDL-induced Arp2 and Arp3 expression. Moreover, Rac-1 siRNA remarkably suppressed ox-LDL-stimulated Arp2 and Arp3 expression. Additionally, CK666 reduced endothelial nitric oxide synthase (eNOS) expression and atherosclerotic lesions in ApoE−/− mice. Collectively, ox-LDL induces endothelial dysfunction by activating LOX-1/Rac-1 signaling and upregulating Arp2/3 complex expression.

Neural Activity-Dependent Regulation of Radial Glial Filopodial Motility Is Mediated by Glial cGMP-Dependent Protein Kinase 1 and Contributes to Synapse Maturation in the Developing Visual System

Radial glia in the developing optic tectum extend highly dynamic filopodial protrusions within the tectal neuropil, the motility of which has previously been shown to be sensitive to neural activity and nitric oxide (NO) release. Using in vivo two-photon microscopy, we performed time-lapse imaging of radial glial cells and measured filopodial motility in the intact albino Xenopus laevis tadpole. Application of MK801 to block neuronal NMDA receptor (NMDAR) currents confirmed a significant reduction in radial glial filopodial motility. This reduction did not occur in glial cells expressing a dominant-negative form of cGMP-dependent protein kinase 1 (PKG1), and was prevented by elevation of cGMP levels with the phosphodiesterase type 5 inhibitor sildenafil. These results suggest that neuronal NMDAR activation results in the release of NO, which in turn modulates PKG1 activation in glial cells to control filopodial motility. We further showed that interfering with the function of the small GTPases Rac1 or RhoA, known to be regulated by PKG1 phosphorylation, decreased motility or eliminated filopodial processes respectively. These manipulations led to profound defects in excitatory synaptic development and maturation of neighboring neurons. Radial glia in the developing brain extend motile filopodia from their primary stalk. Neuronal NMDA receptor activity controls glial motility through intercellular activation of cGMP-dependent protein kinase 1 (PKG1) signaling in glial cells. Manipulating PKG1, Rac1, or RhoA signaling in radial glia in vivo to eliminate glial filopodia or impair glial motility profoundly impacted synaptogenesis and circuit maturation.

Cleavage of Hyaluronan and CD44 Adhesion Molecule Regulate Astrocyte Morphology via Rac1 Signalling.

Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a “stellate”-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of astrocytes.

Abstract 650: Statins Modulate Rac-dependent IL-1 beta Expression to Influence Calcium Composition

Coronary artery disease caused by atherosclerosis is the leading cause of morbidity and mortality in the world. Calcification of atherosclerotic plaque has predictive value in terms of cardiovascular event risk and mortality. The composition of calcium within plaque ( i.e. increased calcium density) has been associated with reduced vulnerability for cardiac events. Therapeutic modulation of atherosclerotic plaque calcium composition may have tremendous potential in terms of reducing event risk. HMG-CoA reductase inhibitors (statins) are a well-established family of medications that reduce the risk of myocardial infarction and are associated with increased plaque calcification, yet the underlying mechanisms of these processes are not well understood. Moreover, statin use can be associated with significant adverse reactions ( i.e. myalgias and new onset diabetes). Our group has identified a Rac-dependent inflammatory pathway that promotes IL-1β-driven atherosclerotic calcification. Using an atherosclerotic-prone mouse model, we found that progressive calcification of atherosclerotic plaque depends on altered Rac signaling and consequent IL-1β expression. Statin-treated primary macrophages expressed higher IL-1β mRNA and secreted more IL-1β protein in response to inflammasome activation by TLR-coupled cholesterol crystal phagocytosis. This statin-induced augmentation of IL-1β production depended on Rac1, as small molecule inhibition of Rac1 and Rac1 gene-deletion abrogated this effect. Statin treatment disrupted the association of Rac1 with its native inhibitor, Rho-GDI, through loss of Rac1 isoprenylation, and as a consequence, statin-treated macrophages demonstrated higher levels of GTP-bound Rac1. This higher activation of Rac1 led to increased NF-κB activation and consequent macrophage IL-1β mRNA expression. ApoE -/- mice placed on high fat diet with statin therapy revealed increases in aortic plaque calcification relative to control mice in association with altered serum IL-1β levels. Defining Rac signaling as a key mediator of statin-dependent mechanisms that modulate plaque calcium composition and consequent plaque stability may lead to novel therapeutic strategies for the prevention of cardiovascular events.

Abstract 655: Rac2 is a Key Modulator of IL-1β -dependent Atherosclerotic Plaque Calcification

The calcium composition of atherosclerotic plaque has predictive value for increased risk of cardiovascular events. Inflammation is associated with atherosclerotic calcification, but the immune signaling that regulates calcium mineralization in plaque is minimally understood. The hematopoietic Rac family member, Rac2, modulates the activation of immune cells and has potential to influence plaque osteogenesis. Both aortic plaque from ApoE -/- mice fed a high fat diet and coronary plaque from patients revealed increased Rac1:Rac2 expression ratios, driven by dynamic Rac2 expression, to be associated with calcified plaque. On high fat diet, Rac2 -/- ApoE -/- mice demonstrated comparable serum cholesterol and plaque burden relative to ApoE -/- mice, but histology identified differences in plaque structure and cellularity. MicroCT and calcium-targeted imaging identified increased atherosclerotic calcification, which was associated with elevated expression of osteogenic transcription factors and was dependent on the hematopoietic compartment. Calcified plaque expressed higher IL-1β mRNA levels, and serum revealed increased IL-1β protein concentrations. Rac2 -/- ApoE -/- macrophages demonstrated increased activation of Rac1 and consequent Rac1-dependent IL-1β secretion. Downstream of Rac1, NF-κB and reactive oxygen species (ROS) signaling drove IL-1β production by increasing IL-1β mRNA expression and caspase1 activation. Cultured mouse aorta smooth muscle cells mineralized calcium in an IL-1β dose-dependent manner, and the enhanced atherosclerotic calcification in vivo was inhibited by IL-1 receptor antagonist, confirming a cause-and-effect relationship. In patients with stable coronary artery disease, high coronary calcium burden was associated with increased serum IL-1β, and patients with combined elevations in calcium and IL-1β had more events driven by higher mortality, reinforcing the relevance of this inflammatory calcification signaling axis to human disease. Therapeutic targeting of IL-1β expression through the balance of Rac activation has potential to impact patient care by modulating atherosclerotic calcification and consequent cardiovascular events.

Distinctive signalling in NK cells during cytotoxicity to the yeast Cryptococcus neoformans

Abstract Introduction Cryptococcus is a fungal pathogen that subverts the adaptive immune response. Fortunately, NK cells are capable of directly recognizing, triggering cytotoxicity and killing Cryptococcus. Although, NK cytotoxicity against tumor and virally infected targets are well studied, less is known about anti-fungal signaling. In this study, we investigated a novel NK cytotoxicity pathway involving Rac, Src family kinases (SFK), and integrins. Methods The involvement of Rac, SFK, integrin-linked kinase (ILK), and integrins were explored using small molecule inhibitors, siRNA knockdown, activated Rac pull down assays, and immunoblots. Results Our studies found that Cryptococcus activated both SFK and Rac signaling pathways. Small molecule inhibitors of SFK and Rac blocked Erk and PI3K activity. siRNA knockdown of Rac also inhibited PI3K activity, suggesting that Rac was an upstream non-canonical activator of PI3K. Moreover, individually Rac and SFK were not sufficient for activation of PI3K. Inhibition of Rac also resulted in a 90% loss of anti-cryptococcal cytotoxicity. Additionally, inhibitors of ILK prevented Rac activation and caused an 80% reduction in cryptococcal killing. Lastly, siRNA knockdown of beta-1 integrins caused an inhibition of ILK activation and 50% reduction in cryptococcal killing. Conclusion We found beta-1 integrins to be a fungal receptor and Rac an activator of PI3K. These findings build a signaling model of anti-fungal killing that is different from the canonical tumor killing model that involves beta-2 integrins and Rac downstream of PI3K. Our model provides various potential therapeutic targets that could enhance fungal clearance mediated by the innate immune system.

Defects in the Fanconi Anemia Pathway in Head and Neck Cancer Cells Stimulate Tumor Cell Invasion through DNA-PK and Rac1 Signaling.

Head and neck squamous cell carcinoma (HNSCC) remains a devastating disease, and Fanconi anemia (FA) gene mutations and transcriptional repression are common. Invasive tumor behavior is associated with poor outcome, but relevant pathways triggering invasion are poorly understood. There is a significant need to improve our understanding of genetic pathways and molecular mechanisms driving advanced tumor phenotypes, to develop tailored therapies. Here we sought to investigate the phenotypic and molecular consequences of FA pathway loss in HNSCC cells. Using sporadic HNSCC cell lines with and without FA gene knockdown, we sought to characterize the phenotypic and molecular consequences of FA deficiency. FA pathway inactivation was confirmed by the detection of classic hallmarks of FA following exposure to DNA cross-linkers. Cells were subjected to RNA sequencing with qRT-PCR validation, followed by cellular adhesion and invasion assays in the presence and absence of DNA-dependent protein kinase (DNA-PK) and Rac1 inhibitors. We demonstrate that FA loss in HNSCC cells leads to cytoskeletal reorganization and invasive tumor cell behavior in the absence of proliferative gains. We further demonstrate that cellular invasion following FA loss is mediated, at least in part, through NHEJ-associated DNA-PK and downstream Rac1 GTPase activity. These findings demonstrate that FA loss stimulates HNSCC cell motility and invasion, and implicate a targetable DNA-PK/Rac1 signaling axis in advanced tumor phenotypes.

Rnd3 as a Novel Target to Ameliorate Microvascular Leakage.

BackgroundMicrovascular leakage of plasma proteins is a hallmark of inflammation that leads to tissue dysfunction. There are no current therapeutic strategies to reduce microvascular permeability. The purpose of this study was to identify the role of Rnd3, an atypical Rho family GTPase, in the control of endothelial barrier integrity. The potential therapeutic benefit of Rnd3 protein delivery to ameliorate microvascular leakage was also investigated.Methods and ResultsUsing immunofluorescence microscopy, Rnd3 was observed primarily in cytoplasmic areas around the nuclei of human umbilical vein endothelial cells. Permeability to fluorescein isothiocyanate–albumin and transendothelial electrical resistance of human umbilical vein endothelial cell monolayers served as indices of barrier function, and RhoA, Rac1, and Cdc42 activities were determined using G‐LISA assays. Overexpression of Rnd3 significantly reduced the magnitude of thrombin‐induced barrier dysfunction, and abolished thrombin‐induced Rac1 inactivation. Depleting Rnd3 expression with siRNA significantly extended the time course of thrombin‐induced barrier dysfunction and Rac1 inactivation. Time‐lapse microscopy of human umbilical vein endothelial cells expressing GFP‐actin showed that co‐expression of mCherry‐Rnd3 attenuated thrombin‐induced reductions in local lamellipodia that accompany endothelial barrier dysfunction. Lastly, a novel Rnd3 protein delivery method reduced microvascular leakage in a rat model of hemorrhagic shock and resuscitation, assessed by both intravital microscopic observation of extravasation of fluorescein isothiocyanate–albumin from the mesenteric microcirculation, and direct determination of solute permeability in intact isolated venules.ConclusionsThe data suggest that Rnd3 can shift the balance of RhoA and Rac1 signaling in endothelial cells. In addition, our findings suggest the therapeutic, anti‐inflammatory potential of delivering Rnd3 to promote endothelial barrier recovery during inflammatory challenge.

Mammary epithelial tubes elongate through MAPK-dependent coordination of cell migration

Mammary branching morphogenesis is regulated by receptor tyrosine kinases (RTKs). We sought to determine how these RTK signals alter proliferation and migration to accomplish tube elongation in mouse. Both behaviors occur but it has been difficult to determine their relative contribution to elongation in vivo, as mammary adipocytes scatter light and limit the depth of optical imaging. Accordingly, we utilized 3D culture to study elongation in an experimentally accessible setting. We first used antibodies to localize RTK signals and discovered that phosphorylated ERK1/2 (pERK) was spatially enriched in cells near the front of elongating ducts, whereas phosphorylated AKT was ubiquitous. We next observed a gradient of cell migration speeds from rear to front of elongating ducts, with the front characterized by both high pERK and the fastest cells. Furthermore, cells within elongating ducts oriented both their protrusions and their migration in the direction of tube elongation. By contrast, cells within the organoid body were isotropically protrusive. We next tested the requirement for proliferation and migration. Early inhibition of proliferation blocked the creation of migratory cells, whereas late inhibition of proliferation did not block continued duct elongation. By contrast, pharmacological inhibition of either MEK or Rac1 signaling acutely blocked both cell migration and duct elongation. Finally, conditional induction of MEK activity was sufficient to induce collective cell migration and ductal elongation. Our data suggest a model for ductal elongation in which RTK-dependent proliferation creates motile cells with high pERK, the collective migration of which acutely requires both MEK and Rac1 signaling.

N‐cadherin adhesion modulates RhoGTPase signaling to promote endothelial barrier integrity

The vascular endothelium is comprised of a monolayer of endothelial cells (ECs) lining the blood vessels, which functions as a semi‐permeable barrier between blood and the surrounding tissue. Vascular Endothelial (VE)‐cadherin, which forms homotypic adhesion between ECs at cell‐cell contacts called adherens junctions (AJs) is the main adhesive molecule that restricts passage of protein rich fluids through the endothelial monolayer. Along with VE‐cadherin, ECs express Neural (N)‐cadherin, which is excluded from EC‐EC junctions and instead forms heterotypic adhesions between ECs and mural cells (such as pericytes or smooth muscle cells) at the abluminal surface of the endothelium. While the role of VE‐cadherin adhesion in regulating endothelial barrier function has been well established, the role of N‐cadherin in ECs remains mainly unknown. Recent evidence suggests that pericytes are critical to maintaining proper endothelial barrier function, however it is still unclear if this is solely due to pericyte recruitment to the endothelium or if N‐cadherin adhesion mediated signaling in ECs plays a role in strengthening the endothelial barrier. Using an EC specific, inducible knockout mouse model developed in our lab, we have shown that deletion of Cdh2 gene (N‐cadherin) results in increased permeability of pulmonary endothelial microvessels, suggesting that N‐cadherin adhesion restricts permeability in the endothelial barrier. To study N‐cadherin adhesion‐mediated signaling in endothelial monolayers, we have utilized surface chemistry techniques to mimic N‐cadherin homophilic interaction in vitro. Covalent attachment of N‐cadherin extracellular domain to an Ni‐NTA surface in an oriented manner induced formation of N‐cadherin adhesions proximal to tricellular junctions and promoted recruitment of VE‐cadherin to AJs. Depletion of N‐cadherin in ECs or denaturation of N‐cadherin extracellular domain abolished this effect, suggesting that N‐cadherin “outside‐in” signaling promotes assembly of VE‐cadherin adhesion. Using microscale biomimetic platforms combined with mass spectroscopy, we isolated and analyzed N‐cadherin complexes. We found that a dual Rac1 and RhoA activator, Trio, is recruited to N‐cadherin adhesion complexes to activate both Rac1 and RhoA signaling at AJs. This was consistent with increased acto‐myosin tension across VE‐cadherin analyzed with a FRET‐based biosensor as well as formation of lamellipodia protrusions above AJs. Furthermore, inhibition of ROCK signaling or depletion of Neuronal navigator 1 (Nav1) abolished recruitment of VE‐cadherin to AJs specifically in cells plated onto N‐cadherin but not gelatin‐coated surfaces. These data cumulatively demonstrate that N‐cadherin promotes assembly of VE‐cadherin adhesion by providing spatial control of RhoA and Rac1 activities through activation of Trio.Support or Funding InformationNIH R01 HL103922

Wild Type p53 Regulates Endothelial Barrier Function by mediating Rac1 signaling and inhibiting RhoA activity

The tumor suppressor and heat shock protein 90 (hsp90) client protein, p53, is a transcription factor which is frequently mutated in human cancers. An emerging body of evidence suggests that p53 also induces strong anti‐inflammatory responses, both in vivo and in vitro. Such activities maintain and preserve vascular barrier function, a key element of lung integrity and defense against endogenous or exogenous insults. We have recently reported that p53 is a major regulator of vascular integrity and endothelial permeability mostly due to its inhibition of the barrier disruptive RhoA signaling. In the present study we further investigated the mechanisms underlying the barrier protective effects of p53. Nutlin‐ or hsp90 inhibitor‐ induced p53 overexpression increased Rac1 activity (GTP‐bound Rac1), a small GTPase with known barrier‐protective effects. Nutlin or hsp90 inhibitor also increased downstream LIMK1/2 phosphorylation/activation as well as cofilin phosphorylation/deactivation thus preventing actin stess fiber formation and promoting cortical actin. When p53 expression was suppressed by siRNA, the opposite effects were observed. Hsp90 inhibitor (AUY‐922)‐induced p53 overexpression also caused an induction of the RhoA negative regulator p190Rho‐GAP that led to inhibition of RhoA activity as well as inhibition of downstream MLC phosphorylation. Real time measurements of endothelial barrier function by continuous monitoring of transendothelial electrical resistance (TER) of human lung microvascular endothelial monolayers revealed a prominent role of p53 in endothelial barrier regulation. Downregulation of p53 by pifithrin attenuated AUY‐922‐induced barrier enhancement, whereas siRNA‐mediated p190Rho‐GAP silencing led to an enhanced LPS‐induced decrease in TER. In conclusion, we propose that p53 regulates vascular barrier function by manipulating the balance of the actin remodeling Rac1 and RhoA pathways.Support or Funding InformationThis study was supported by National Heart, Lung, and Blood Institute Grants HL101902 and HL93460.

Temporal Rac1 – HIF-1 crosstalk modulates hypoxic survival of aged neurons

Neurodegenerative diseases are frequently associated with hypoxic conditions. During hypoxia the neuronal cytoskeleton is rapidly reorganized and such abnormalities are directly linked to adverse outcomes. Besides their roles as master regulators of the cytoskeleton, the Rho GTPases are also involved in cellular processes stimulated by hypoxic stress. We investigated the contribution of Rac1-mediated signaling to hypoxic responses of mature neurons using primary cortical cells cultured for 17 days in vitro. We show Rac1 is both upregulated and activated during hypoxia. Pharmacological inhibition of Rac1, but not RhoA, completely abrogated hypoxic HIF-1α stabilization and expression of the HIF-1 targets VEGF and GLUT1. Furthermore activity of JNK and GSK3β were also highly dependent on Rac1 activity and biphasic effects were observed after 6 and 24h of exposure. Notably, inhibition of either pathway suppressed HIF-1α accumulation. Although inhibition of Rac1 did not affect neuronal viability during acute exposure cell death was strongly induced after 24h revealing a time-dependent effect of Rac1 signaling on survival. Thus hypoxia-activated Rac1 is critical for neuronal HIF-1α stabilization and survival during oxygen deprivation via integration of complex signaling cascades.

Deletion of Rac1GTPase in the Myeloid Lineage Protects against Inflammation-Mediated Kidney Injury in Mice.

Macrophage-mediated inflammation has been implicated in various kidney diseases. We previously reported that Rac1, a Rho family small GTP-binding protein, was overactivated in several chronic kidney disease models, and that Rac1 inhibitors ameliorated renal injury, in part via inhibition of inflammation, but the detailed mechanisms have not been clarified. In the present study, we examined whether Rac1 in macrophages effects cytokine production and the inflammatory mechanisms contributing to kidney derangement. Myeloid-selective Rac1 flox control (M-Rac1 FC) and knockout (M-Rac1 KO) mice were generated using the cre-loxP system. Renal function under basal conditions did not differ between M-Rac1 FC and KO mice. Accordingly, lipopolysaccharide (LPS)-evoked kidney injury model was created. LPS elevated blood urea nitrogen and serum creatinine, enhanced expressions of kidney injury biomarkers, Kim-1 and Ngal, and promoted tubular injury in M-Rac1 FC mice. By contrast, deletion of myeloid Rac1 almost completely prevented the LPS-mediated renal impairment. LPS triggered a marked induction of macrophage-derived inflammatory cytokines, IL-6 and TNFα, in M-Rac1 FC mice, which was accompanied by Rac1 activation, stimulation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and reactive oxygen species overproduction. These changes were inhibited in M-Rac1 KO mice. LPS evoked F4/80-positive macrophages accumulation in the kidney, which was not affected by myeloid Rac1 deficiency. We further tested the role of Rac1 signaling in cytokine production using macrophage cell line, RAW264.7. Exposure to LPS increased IL-6 and TNFα mRNA expression. The LPS-driven cytokine induction was dose-dependently blocked by the Rac1 inhibitor EHT1864, NADPH oxidase inhibitor diphenyleneiodonium, and NF-κB inhibitor BAY11-7082. In conclusion, genetic ablation of Rac1 in the myeloid lineage protected against LPS-induced renal inflammation and injury, by suppressing macrophage-derived cytokines, IL-6 and TNFα, without blocking recruitment. Our data suggest that Rac1 in macrophage is a novel target for the treatment of kidney disease through inhibition of cytokine production.

Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration.

The small GTPase Rac1 has been implicated in the formation and dissemination of tumours. Upon activation by guanine nucleotide exchange factors (GEFs), Rac1 associates with a variety of proteins in the cell thereby regulating various functions, including cell migration. However, activation of Rac1 can lead to opposing migratory phenotypes raising the possibility of exacerbating tumour progression when targeting Rac1 in a clinical setting. This calls for the identification of factors that influence Rac1-driven cell motility. Here we show that Tiam1 and P-Rex1, two Rac GEFs, promote Rac1 anti- and pro-migratory signalling cascades, respectively, through regulating the Rac1 interactome. In particular, we demonstrate that P-Rex1 stimulates migration through enhancing the interaction between Rac1 and the actin-remodelling protein flightless-1 homologue, to modulate cell contraction in a RhoA-ROCK-independent manner.