Leukemia-associated Rho Guanine Nucleotide Exchange Research Articles

Small chemical compounds Y16 and Rhosin can inhibit calcium sensitization pathway in vascular smooth muscle cells of spontaneously hypertensive rats

The small-molecule compounds Y16 and Rhosin can inhibit the activation of leukemia-associated Rho guanine nucleotide exchange factor (LARG) and small G-protein RhoA, respectively, in breast cancer cells and inhibit their growth and migration. However, it remains unclear whether they have inhibitory effects on the vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs). Primary cultured VSMCs from SHRs were treated with different concentrations of Y16 or Y16 plus Rhosin for 24h, followed by 10-min stimulation with 10-7M angiotensin II (Ang II). The cells were then harvested, and the total protein was extracted. The co-immunoprecipitation method, Western blot analysis, and MTT assay were performed to determine the LARG-RhoA interaction, the protein levels of RhoA and MYPT1, and cell viability, respectively. Y16 dose-dependently inhibited the LARG-RhoA complex formation induced by Ang II. With 50μM of Y16, the effect of inhibition was statistically significant. Y16 also reduced the formation of phospho-MYPT1 stimulated by Ang II. With 5μM of Y16, the inhibitory effect was statistically significant. When 25μM of Y16 and 25μM of Rhosin were combined, the inhibitory effect on LARG-RhoA interaction was statistically significant. When Y16 and Rhosin were combined, a significantly reduced concentration could effectively inhibit MYPT1 phosphorylation (2.5μM compared with 5μM for Y16 alone). Treating SHR VSMCs with Y16 can suppress the activation of LARG, prevent LARG binding to RhoA, and decrease the phosphorylation of MYPT1, thus weakening the activation of the calcium (Ca2+) sensitization pathway in SHR VSMCs.

Leukemia-Associated Rho Guanine Nucleotide Exchange Factor and Ras Homolog Family Member C Play a Role in Glioblastoma Cell Invasion and Resistance

Glioblastoma (GBM) is the most common primary malignant brain cancer in adults. A hallmark of GBM is aggressive invasion of tumor cells into the surrounding normal brain. Both the current standard of care and targeted therapies have largely failed to specifically address this issue. Therefore, identifying key regulators of GBM cell migration and invasion is important. The leukemia-associated Rho guanine nucleotide exchange factor (LARG) has previously been implicated in cell invasion in other tumor types; however, its role in GBM pathobiology remains undefined. Herein, we report that the expression levels of LARG and ras homolog family members C (RhoC), and A (RhoA) increase with glial tumor grade and are highest in GBM. LARG and RhoC protein expression is more prominent in invading cells, whereas RhoA expression is largely restricted to cells in the tumor core. Knockdown of LARG by siRNA inhibits GBM cell migration invitro and invasion exvivo in organotypic brain slices. Moreover, siRNA-mediated silencing of RhoC suppresses GBM cell migration invitro and invasion exvivo, whereas depletion of RhoA enhances GBM cell migration and invasion, supporting a role for LARG and RhoC in GBM cell migration and invasion. Depletion of LARG increases the sensitivity of GBM cells to temozolomide treatment. Collectively, these results suggest that LARG and RhoC may represent unappreciated targets to inhibit glioma invasion.

Regulation of neurite morphogenesis by interaction between R7 regulator of G protein signaling complexes and G protein subunit Gα13

The R7 regulator of G protein signaling family (R7-RGS) critically regulates nervous system development and function. Mice lacking all R7-RGS subtypes exhibit diverse neurological phenotypes, and humans bearing mutations in the retinal R7-RGS isoform RGS9-1 have vision deficits. Although each R7-RGS subtype forms heterotrimeric complexes with Gβ5 and R7-RGS-binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of Gi/o α-subunits, several neurological phenotypes of R7-RGS knock-out mice are not readily explained by dysregulated Gi/o signaling. Accordingly, we used tandem affinity purification and LC-MS/MS to search for novel proteins that interact with R7-RGS heterotrimers in the mouse brain. Among several proteins detected, we focused on Gα13 because it had not been linked to R7-RGS complexes before. Split-luciferase complementation assays indicated that Gα13 in its active or inactive state interacts with R7-RGS heterotrimers containing any R7-RGS isoform. LARG (leukemia-associated Rho guanine nucleotide exchange factor (GEF)), PDZ-RhoGEF, and p115RhoGEF augmented interaction between activated Gα13 and R7-RGS heterotrimers, indicating that these effector RhoGEFs can engage Gα13·R7-RGS complexes. Because Gα13/R7-RGS interaction required R7BP, we analyzed phenotypes of neuronal cell lines expressing RGS7 and Gβ5 with or without R7BP. We found that neurite retraction evoked by Gα12/13-dependent lysophosphatidic acid receptors was augmented in R7BP-expressing cells. R7BP expression blunted neurite formation evoked by serum starvation by signaling mechanisms involving Gα12/13 but not Gαi/o These findings provide the first evidence that R7-RGS heterotrimers interact with Gα13 to augment signaling pathways that regulate neurite morphogenesis. This mechanism expands the diversity of functions whereby R7-RGS complexes regulate critical aspects of nervous system development and function.

PS 10-04 HYPOTENSIVE EFFECT OF LOSARTAN THROUGH SUPPRESSING LEUKEMIA-ASSOCIATED RhoGEF EXPRESSION IN VASCULAR SMOOTH MUSCLE OF SPONTANEOUSLY HYPERTENSIVE RATS

Objective: To elucidate the mechanism that chronic blood pressure (BP) reduction caused by losartan is through the suppression of leukemia-associated Rho guanine nucleotide exchange factors (LARG) expression in vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs). Design and Method: Four-week-old Wistar-Kyoto (WKY) rats and SHRs were treated with Losartan (20 mg/kg/day) for 8 weeks. BP was measured by the tail-cuff method. At 12 weeks, isometric contraction of rat aortic rings was measured with a force transducer and recorder. The target Rho guanine nucleotide exchange factors (RhoGEFs) messenger RNA (mRNA) and protein levels, and the myosin phosphatase target subunit 1 (MYPT-1) phosphorylation extent in aorta were determined using quantitative real-time polymerase chain reaction (qPCR) assay and Western blot analysis, respectively. Results: BP of four-week-old SHRs did not differ from that of age-matched WKY rats, whereas BP increased in twelve-week-old SHRs. Losartan treatment reduced BP in both rats’ strains, with greater extent in SHRs. The contractile force of aortic rings from SHRs treated with losartan was significantly lower than that from non-treated SHRs in response to angiotensin II. LARG protein expression was significantly higher in non-treated SHRs compared to WKY rats. After losartan treatment, both LARG protein level and phosphorylation extent of MYPT-1 were more suppressed in SHRs than in WKY rats. Conclusions: The study shows that chronic reduction of BP by losartan in SHRs is through suppressing LARG expression and MYPT-1 phosphorylation of VSMCs. Administration of losartan at early age of SHRs may help prevent hypertension development.

Ligand-dependent serum response factor activation by the human CC chemokine receptors CCR2a and CCR2b is mediated by G proteins of the Gq family.

Expression of the human CCR2 receptors, CCR2a and CCR2b, in mammalian cells results in ligand-dependent changes in the activity of multiple cellular signal transduction pathways, mediated in most cases by pertussis toxin-sensitive heterotrimeric G proteins of the Gi/o subfamily. In addition, CCR2a and CCR2b receptors have been shown to couple to Gq family members, triggering the canonical activation of phospholipase Cβ isoenzymes. Activation of pertussis toxin-insensitive Gq proteins by cell-surface receptors is not only coupled to activation of phospholipase isoenzymes but also to Rho guanine nucleotide exchange factors, which in turn mediate activation of the Rho GTPases. Activated Rho GTPases regulate numerous cellular functions, including the organization of the actin cytoskeleton and gene transcription, such as the transcription factor serum response factor. These findings prompted us to investigate whether CCR2a and/or CCR2b stimulate serum response factor activity. The results presented herein demonstrate that stimulation of human CCR2a- or CCR2b-expressing COS-7 cells caused a vigorous induction of serum response factor activity. This effect was specifically mediated by Gq and/or G14, as well as Rho A and/or a closely related Rho GTPase. Furthermore, the stimulatory effect of CCR2a and CCR2b and Gαq was sensitive to coexpression of the Gαq-interacting leukemia-associated Rho guanine nucleotide exchange factor. The findings of the work indicate a role for Gαq and/or Gα14 and in CCR2a/CCR2b-stimulated Rho A GTPase-mediated serum response factor activation and introduce a noncanonical pathway activated by CCR2 receptors by coupling to Gq proteins.

The RhoA Guanine Nucleotide Exchange Factor, LARG, Mediates ICAM-1–Dependent Mechanotransduction in Endothelial Cells To Stimulate Transendothelial Migration

RhoA-mediated cytoskeletal rearrangements in endothelial cells (ECs) play an active role in leukocyte transendothelial cell migration (TEM), a normal physiological process in which leukocytes cross the endothelium to enter the underlying tissue. Although much has been learned about RhoA signaling pathways downstream from ICAM-1 in ECs, little is known about the consequences of the tractional forces that leukocytes generate on ECs as they migrate over the surface before TEM. We have found that after applying mechanical forces to ICAM-1 clusters, there is an increase in cellular stiffening and enhanced RhoA signaling compared with ICAM-1 clustering alone. We have identified that leukemia-associated Rho guanine nucleotide exchange factor (LARG), also known as Rho GEF 12 (ARHGEF12) acts downstream of clustered ICAM-1 to increase RhoA activity, and that this pathway is further enhanced by mechanical force on ICAM-1. Depletion of LARG decreases leukocyte crawling and inhibits TEM. To our knowledge, this is the first report of endothelial LARG regulating leukocyte behavior and EC stiffening in response to tractional forces generated by leukocytes.

Ras homolog gene family member A (RHOA); leukemia-associated Rho guanine nucleotide exchange factor (LARG)

Ras homolog gene family member A (RHOA); leukemia-associated Rho guanine nucleotide exchange factor (LARG)

Hepatocyte nuclear factor (HNF)-1β and its physiological importance in endometriosis

Endometriosis is associated with pelvic pain and female infertility. Endometriosis induces inflammation and is vulnerable to oxidative stress damage. To update and summarize the literature concerning the mechanisms that serve to protect genomic DNA from the oxidative damage, the present study reviews the English-language literature for biochemical studies on the transcription factor hepatocyte nuclear factor (HNF)-1β target genes. Findings demonstrated that retrograde flow of the menstrual blood might give rise to endometriosis. Iron may have a significant impact on endometriosis gene expression. HNF-1β regulates tissue-specific gene expression in endometriosis, as well as the expression of several genes, including CD44v9, which binds several molecules, including hyaluronan, epidermal growth factor receptor (EGFR), leukemia-associated Rho-guanine nucleotide exchange factor (LARG), IQ motif containing GTPase activating protein 1 (IQGAP1), macrophage migration inhibitory factor (MIF), major histocompatibility complex, class II invariant chain (CD74), cystine transporter subunit (xCT), Fas and extracellular matrix (ECM) proteins. The CD44v9 system is involved in cell migration, growth, survival, anti-apoptosis, immune response and anti-oxidative stress through maintaining higher levels of antioxidants. HNF-1β may serve to alleviate damage and promote survival of cells experiencing stress by upregulating antioxidant protein expression. This review expands current knowledge on the molecular mechanisms underlying the oxidative stress protection provided by HNF-1β and provides evidence that elevated HNF-1β activity might be associated with the CD44v9-dependent signaling cascades.

Differential baseline expression and angiotensin II stimulation of leukemia-associated RhoGEF in vascular smooth muscle cells of spontaneously hypertensive rats

PurposeStudies to explore angiotensin II (Ang II) and its downstream signaling pathways via Rho guanine nucleotide exchange factors (RhoGEFs) and RhoA signaling are crucial to understanding the mechanisms of smooth muscle contraction leading to hypertension. This study aimed to investigate the Ang II–induced expression of RhoGEFs in vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs) and to identify the possible regulator associated with hypertension.MethodsCultured VSMCs of the aorta from SHRs and Wistar-Kyoto (WKY) rats were treated with or without Ang II or Ang II plus Ang II type 2 receptor antagonists. The expression levels of RhoGEF messenger RNA (mRNA) and protein were determined. To evaluate the changes of aortic ring contractile force in response to Ang II, a nonviral carrier system was adopted to deliver the leukemia-associated RhoGEF (LARG) small interfering RNA via nanoparticles into aortic rings.ResultsThe baseline mRNA levels of three RhoGEFs in cultured VSMCs of WKY rats did not increase with age, but they were significantly higher in 12-week-old SHRs than in 5-week-old SHRs. Expression levels of LARG mRNA were higher in SHRs than in age-matched WKY rats. The baseline LAGR protein of 12-week-old SHRs was about four times higher than that of WKY rats of the same age. After Ang II–stimulation, LAGR protein expression was significantly increased in 12-week-old WKY rats but remained unchanged in 12-week-old SHRs. LARG small interfering RNA was successfully delivered into aortic rings using nanoparticles. LARG knockdown resulted in 12-week-old SHRs showing the greatest reduction in aortic ring contraction.ConclusionThere were differences in age-related RhoGEF expression at baseline and in response to Ang II–stimulation between SHRs and WKY rats in this study. Nanotechnology can assist in studying the silencing of LARG in tissue culture. The findings of this study indicate that LARG gene expression may be associated with the genesis of hypertension in SHRs.

The Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (LARG) Is Essential for Survival and Proliferation of AML1-ETO Associated AML Cells

Abstract 923AML1-ETO (AE), the product of translocation 8;21, is the most frequently observed fusion gene in acute myeloid leukemia (AML). Although AE is not sufficient to induce leukemia by itself, it endows a significant survival and growth advantage to hematopoietic progenitors. In a search of various AML microarray databases, we found that the Leukemia-Associated Rho Guanine nucleotide exchange factor (LARG) gene expression is consistently upregulated in samples from AE-positive AML patients compared to other types of AML. This observation was confirmed in an independent cohort of AML samples by qPCR and Western Blot analysis, and in a pre-leukemia cell model generated by AE expression in CD34+ human cord blood (CB) cells compared with CB cells transduced with the MLL-AF9 (MA9) fusion gene or control retroviral vector. ChIP-PCR assays further indicate that AE directly binds to the LARG regulatory region through cis-elements containing AML1 (Runx1) binding sites. LARG, a RhoA GEF, can activate the RhoA pathway which is important for interaction between cells and their environment. We found that the AE cell lines were more adhesive to fibronectin than the MA9 cell lines. When we used lentiviral vectors expressing shRNA to suppress LARG expression in AE+ pre-leukemic cells, the cells showed a significant decrease in adhesion to fibronectin. In addition, knockdown of LARG also significantly interfered with the growth of AE cells in that the shRNA transduced cells displayed a competitive disadvantage relative to non-transduced or control-transduced cells in a proliferation assay. Cell cycle analysis revealed that LARG knockdown induced cell cycle exit, while Annexin V staining showed that LARG suppression promoted apoptosis of AE cells. Because LARG is a well-known guanine nucleotide exchange factor (GEF) for RhoA GTPase, we examined the downstream signaling events of the LARG/Rho axis, including phosphorylation of MLC and AURORA, targets of Rho-associated protein kinase (ROCK). pMLC and pAURORA were significantly down-regulated upon knockdown of LARG. Phosphorylation of Stat3, another protein downstream of RhoA signaling important for the proliferation and survival of myeloid cells, was also decreased upon LARG knockdown. These findings suggest that LARG is a transcriptional target of the AML1-ETO fusion protein and the LARG-RhoA signaling pathway plays an essential role in the proliferation and survival of AE cells. The LARG/RhoA pathway may serve as a new therapeutic target in t(8;21) AML. Disclosures:No relevant conflicts of interest to declare.

Role of Hyaluronan-Mediated CD44 Signaling in Head and Neck Squamous Cell Carcinoma Progression and Chemoresistance

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy that may involve the oral cavity, pharynx, larynx, and paranasal sinuses. The mechanisms of tumor progression underlying the clinical behavior of HNSCC remain unclear. CD44 comprises a family of transmembrane receptors that can give rise to multiple CD44 variant isoforms. Hyaluronan (HA), a major extracellular matrix component is the primary ligand for CD44 receptors. HA and CD44 signaling play an important role in HNSCC progression. Several CD44 variant isoforms (including v3-, v6-, and v10-containing isoforms) are associated with advanced disease, possibly through unique growth factor interactions with binding domains in the inserted variant regions of the cytoplasmic domain of CD44. In HNSCC, HA mediates the formation of a complex including CD44 and the epidermal growth factor receptor (EGFR) which is overexpressed in a large proportion of HNSCCs. Downstream effectors under EGFR regulation are activated, promoting promote cell growth and tumor survival. The leukemia-associated Rho-guanine nucleotide exchange factor (LARG) also associates with CD44 and EGFR to promote several Ras and RhoA pathway effectors, leading to cell migration, growth, and tumor survival. The secretion of matrix metalloproteinases, necessary for tumor cell invasion, is also regulated by these HA/CD44-mediated pathways. Finally, EGFR-mediated pathways play major roles in the HA/CD44 promotion of chemoresistance in HNSCC. Understanding HA/CD44-mediated signaling pathways may lead to improved treatment of HNSCC.

Conformational change upon ligand binding and dynamics of the PDZ domain from leukemia‐associated Rho guanine nucleotide exchange factor

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) is a RhoA-specific guanine nucleotide exchange factor (GEF) that can activate RhoA. The PDZ (PSD-95/Disc-large/ZO-1 homology) domain of LARG interacts with membrane receptors, which can relay extracellular signals to RhoA signal transduction pathways. Until now there is no structural and dynamic information about these interactions. Here we report the NMR structures of the LARG PDZ in the apo form and in complex with the plexin-B1 C-terminal octapeptide. Unobservable resonances of the residues in betaB/betaC and betaE/alphaB loops in apo state were observed in the complex state. A distinct region of the binding groove in the LARG PDZ was found to undergo conformational change compared with other PDZs. Analysis of the (15)N relaxation data using reduced spectral density mapping shows that the apo LARG PDZ (especially its ligand-binding groove) is flexible and exhibits internal motions on both picosecond to nanosecond and microsecond to millisecond timescales. Mutagenesis and thermodynamic studies indicate that the conformation of the betaB/betaC and betaE/alphaB loops affects the PDZ-peptide interaction. It is suggested that the conformational flexibility could facilitate the change of structures upon ligand binding.

G12-G13–LARG–mediated signaling in vascular smooth muscle is required for salt-induced hypertension

The tone of vascular smooth muscle cells is a primary determinant of the total peripheral vascular resistance and hence the arterial blood pressure. Most forms of hypertension ultimately result from an increased vascular tone that leads to an elevated total peripheral resistance. Regulation of vascular resistance under normotensive and hypertensive conditions involves multiple mediators, many of which act through G protein-coupled receptors on vascular smooth muscle cells. Receptors that mediate vasoconstriction couple with the G-proteins G(q)-G11 and G12-G13 to stimulate phosphorylation of myosin light chain (MLC) via the Ca2+/MLC kinase- and Rho/Rho kinase-mediated signaling pathways, respectively. Using genetically altered mouse models that allow for the acute abrogation of both signaling pathways by inducible Cre/loxP-mediated mutagenesis in smooth muscle cells, we show that G(q)-G11-mediated signaling in smooth muscle cells is required for maintenance of basal blood pressure and for the development of salt-induced hypertension. In contrast, lack of G12-G13, as well as of their major effector, the leukemia-associated Rho guanine nucleotide exchange factor (LARG), did not alter normal blood pressure regulation but did block the development of salt-induced hypertension. This identifies the G12-G13-LARG-mediated signaling pathway as a new target for antihypertensive therapies that would be expected to leave normal blood pressure regulation unaffected.

Genetic variants in the leukemia-associated Rho guanine nucleotide exchange factor (ARHGEF12) gene are not associated with T2DM and related parameters in Caucasians (KORA study)

The aim of our study was to determine the variant pattern of the leukemia-associated Rho guanine nucleotide exchange factor (LARG, or ARHGEF12) gene and investigate whether LARG variants are associated with diabetes mellitus type 2 (T2DM), the metabolic syndrome (MetS), or related parameters such as insulin sensitivity in German Caucasians. We analyzed single nucleotide polymorphisms (SNPs) in the LARG gene in the 55-74-year-old individuals of the population-based German Caucasian Cooperative Health Research in the region of Augsberg (KORA) survey 4 (S4). Sequencing of Tyr1306Cys, which was of functional relevance in Pima Indians, in 48 randomly selected individuals and genotyping of 11 additional LARG SNPs in 1653 subjects were performed. Four linkage disequilibrium (LD) blocks (r(2)> or =0.8) were established and each block was statistically analyzed for association with metabolic traits. The association with T2DM and the MetS was analyzed by logistic regression in 1462 subjects, and HOMA-IR (homeostasis model assessment of insulin resistance) as a measure of insulin sensitivity was analyzed by the Kruskal-Wallis test in 1346 fasting subjects. The polymorphism Tyr1306Cys, which was significantly associated with insulin sensitivity in Pima Indians, was not found in the KORA S4 population. Statistical analysis yielded no significant associations (P>0.05) between the analyzed LARG variants and T2DM, the MetS, or related parameters such as insulin sensitivity. Caucasian individuals and Pima Indians differ in their genetic variance pattern in the LARG gene region. There is no evidence in the Caucasian KORA study that variants of the LARG gene confer susceptibility for T2DM, insulin sensitivity, or the MetS.

Identification of a Molecular Target for the Yersinia Protein Kinase A

Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject bacterial effector proteins directly into the host cytosol. One of these effectors, the Yersinia serine/threonine protein kinase YpkA, is an essential virulence determinant involved in host actin cytoskeletal rearrangements and in inhibition of phagocytosis. Here we report that YpkA inhibits multiple Galphaq signaling pathways. The kinase activity of YpkA is required for Galphaq inhibition. YpkA phosphorylates Ser47, a key residue located in the highly conserved diphosphate binding loop of the GTPase fold of Galphaq. YpkA-mediated phosphorylation of Ser47 impairs guanine nucleotide binding by Galphaq. Y. pseudotuberculosis expressing wild-type YpkA, but not a catalytically inactive YpkA mutant, interferes with Galphaq-mediated signaling pathways. Identification of a YpkA-mediated phosphorylation site in Galphaq sheds light on the contribution of the kinase activity of YpkA to Yersinia pathogenesis.

A Functional Tyr1306Cys Variant in LARG Is Associated With Increased Insulin Action in Vivo

Diminished insulin sensitivity is a characteristic feature of type 2 diabetes. Inhibition of insulin action, resulting in reduced skeletal muscle glucose uptake, is mediated in part through stimulation of RhoA activity. One regulator of RhoA activity is leukemia-associated Rho guanine nucleotide exchange factor (LARG). The LARG gene maps to a region on chromosome 11q23-24 that shows genetic linkage to BMI and type 2 diabetes in Pima Indians. Because of its role in RhoA activation, the LARG gene was analyzed as a positional candidate gene for this linkage. Sequencing of the LARG gene and genotyping of variants identified several polymorphisms that were associated with in vivo rates of insulin-mediated glucose uptake, at both physiological and maximally stimulating insulin concentrations, among 322 nondiabetic Pima Indians who had undergone a hyperinsulinemic-euglycemic clamp. The strongest association with rate of glucose uptake was found with a Tyr1306Cys polymorphism (P < 0.0001, adjusted for age, sex, percent body fat, and nuclear family membership). In transient transfection studies in NIH3T3 cells, the LARG(Cys1306) protein had reduced activity compared with LARG(Tyr1306) protein (P < 0.05). We propose that the Tyr1306Cys substitution in LARG, through its differential activation of RhoA, increases insulin sensitivity in nondiabetic Pima Indians.

Influence of simulated microgravity on the activation of the small GTPase Rho involved in cytoskeletal formation – molecular cloning and sequencing of bovine leukemia-associated guanine nucleotide exchange factor

BackgroundThe irregular formation of cytoskeletal fibers in spaceflown experimental cells has been observed, but the disorganization process of fibers is still poorly understood. It is well known that the activation of the small GTPase Rho leads to actin stress fibers assembly. This study was performed to evaluate the effect of simulated microgravity on the activation of Rho that is involved in actin fiber remodeling in cells.ResultsClinorotation influences actin fiber remodeling and its related signaling pathways that involve the small GTPase Rho. Actin stress fiber remodeling was significantly inhibited to a greater extent in cells cultured under clinorotation than in static cultured cells. From the gene and protein expression analyses, we found that the expression level of leukemia-associated Rho guanine nucleotide exchange factor (LARG), which activates Rho, was downregulated under clinorotation. Moreover, we identified the full-length LARG cDNA. The amount of GTP-bound RhoA, that is, the active form of RhoA, decreased under this condition.ConclusionThe activation of the small GTPase Rho was influenced by simulated microgravity generated by a three-dimensional (3D) clinostat. Furthermore, the full-length cDNA of bovine LARG, a member of the Rho guanine nucleotide exchange factor (GEF) family, was identified, and its gene expression was observed to be downregulated under clinorotation. This downregulation subsequently resulted in the repression of RhoA activation. These results indicated that the disorganization of the actin fibers was caused by the inhibition of Rho activation by 3D clinorotation.

Angiotensin II Up-Regulates the Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (RhoGEF), a Regulator of G Protein Signaling Domain-Containing RhoGEF, in Vascular Smooth Muscle Cells

In vascular smooth muscle, stimulation of heterotrimeric G protein-coupled receptors (GPCRs) by various contractile agonists activates intracellular signaling molecules to result in an increase in cytosolic Ca2+ and the subsequent phosphorylation of myosin light chain (MLC) by Ca2+/calmodulin-dependent MLC kinase. In addition, a portion of agonist-induced contraction is partially mediated by the Ca2+-independent activation of the small G protein RhoA and a downstream target, Rho-kinase. The activation of RhoA is controlled by several regulatory proteins, including guanine nucleotide exchange factors (GEFs). GEFs activate RhoA by promoting the release of GDP and then facilitating the binding of GTP. There are three Rho-specific GEFs (RhoGEFs) in vascular smooth muscle that contain a binding domain [regulator of G protein signaling (RGS) domain] capable of linking GPCRs to RhoA activation: PDZ-RhoGEF, leukemia-associated RhoGEF (LARG), and p115RhoGEF. We hypothesized that RGS domain-containing RhoGEFs, especially LARG, participate in linking GPCR to RhoA activation in vascular smooth muscle. We observed that angiotensin II up-regulates LARG via the AT1 receptor, and this up-regulation is signaled via the phosphatidylinositol 3-kinase pathway. Furthermore, angiotensin II treatment caused a small, but significant, increase in the component of contractile responses sensitive to Rho-kinase antagonism. These observations support the hypothesis that RhoGEFs, particularly LARG, participate in linking GPCR to RhoA activation in vascular smooth muscle.

Larger than Dbl: new structural insights into RhoA activation

Dbl homology (DH) domains are almost always followed immediately by pleckstrin homology (PH) domains in Dbl family proteins, and these DH-PH fragments directly activate GDP-bound Rho GTPases by catalyzing the exchange of GDP for GTP. New crystal structures of the DH-PH domains from leukemia-associated Rho guanine nucleotide exchange factor (RhoGEF) and PDZ-RhoGEF bound to RhoA reveal how DH-PH domains cooperate to specifically activate Rho GTPases.

Characterization of leukemia-associated Rho guanine nucleotide exchange factor (LARG) expression during murine development.

LARG (leukemia-associated Rho guanine nucleotide exchange factor, ARHGEF12) was originally identified as a fusion partner of the MLL gene at 11q23 in human acute myeloid leukemia. We have previously demonstrated that the LARG protein activates RhoA, a member of the Rho family of small GTPases, by catalyzing the exchange of GTP for GDP. Experiments in Drosophila melanogaster have implicated RhoA and its regulators in a spectrum of developmental processes-including gastrulation, neurite outgrowth, and epidermal morphogenesis; however, the role of these genes during mammalian development is incompletely understood. Herein, we investigate the expression of the murine LARG homologue during embryogenesis and in adult animals, by a combination of mRNA in situ hybridization and immunohistochemical detection of the LARG protein. We observe that LARG transcript and protein are undetectable prior to embryonic day 14. Beginning at this stage, LARG is expressed in the skin, intestinal epithelium, and smooth muscle layers of the intestine, bronchi, and vasculature. This specific distribution is maintained at later stages of development and into adulthood. Finally, we demonstrate colocalization of the LARG protein with the insulin-like growth factor-I (IGF-1) receptor, suggesting a potential physiologic role for LARG as an activator of RhoA in response to IGF-1.