Increased RhoA Activity Research Articles

Inhibition of Rho via Arg and p190RhoGAP in the Postnatal Mouse Hippocampus Regulates Dendritic Spine Maturation, Synapse and Dendrite Stability, and Behavior

The RhoA (Rho) GTPase is a master regulator of dendrite morphogenesis. Rho activation in developing neurons slows dendrite branch dynamics, yielding smaller, less branched dendrite arbors. Constitutive activation of Rho in mature neurons causes dendritic spine loss and dendritic regression, indicating that Rho can affect dendritic structure and function even after dendrites have developed. However, it is unclear whether and how endogenous Rho modulates dendrite and synapse morphology after dendrite arbor development has occurred. We demonstrate that a Rho inhibitory pathway involving the Arg tyrosine kinase and p190RhoGAP is essential for synapse and dendrite stability during late postnatal development. Hippocampal CA1 pyramidal dendrites develop normally in arg-/- mice, reaching their mature size by postnatal day 21 (P21). However, dendritic spines do not undergo the normal morphological maturation in these mice, leading to a loss of hippocampal synapses and dendritic branches by P42. Coincident with this synapse and dendrite loss, arg-/- mice exhibit progressive deficits in a hippocampus-dependent object recognition behavioral task. p190RhoGAP localizes to dendritic spines, and its activity is reduced in arg-/- hippocampus, leading to increased Rho activity. Although mutations in p190rhogap enhance dendritic regression resulting from decreased Arg levels, reducing gene dosage of the Rho effector ROCKII can suppress the dendritic regression observed in arg-/- mice. Together, these data indicate that signaling through Arg and p190RhoGAP acts late during synaptic refinement to promote dendritic spine maturation and synapse/dendrite stability by attenuating synaptic Rho activity.

Rac1 Signaling Stimulates N-cadherin Expression, Mesenchymal Condensation, and Chondrogenesis

The molecular mechanisms controlling differentiation of mesenchymal precursor cells into chondrocytes (chondrogenesis) are not completely understood. We have recently shown that the small GTPase RhoA inhibits this process. Here we demonstrate that a different Rho GTPase family member, Rac1, promotes chondrogenesis. Pharmacological inhibition of Rac1 expression in micromass culture resulted in reduced mRNA levels of the chondrogenic markers collagen II and aggrecan, and decreased accumulation of glycosaminoglycans. Expression of the essential chondrogenic transcription factors Sox9, Sox5, and Sox6 was also reduced upon inhibition of Rac1 signaling. In contrast, overexpression of Rac1 in the chondrogenic ATDC5 cell line increased mRNA transcripts of Sox9, 5, and 6, collagen II, and aggrecan. Inhibition of Rac1 resulted in a reduction in the number, size, and organization of cellular condensations and decreased expression of N-cadherin. Overexpression of Rac1 resulted in an increase in N-cadherin expression levels. Furthermore, genetic ablation of Rac1 in primary micromass cultures resulted in reduced expression of chondrogenic markers. Additionally, we provide evidence that Cdc42 also promotes chondrogenesis. Overexpression of Cdc42 in ATDC5 cells resulted in increased expression of Sox5, Sox9, and collagen II but not Sox6, aggrecan, or N-cadherin. Therefore, we demonstrate that Rac1 and Cdc42 are positive regulators of chondrogenesis, but act at least in part through different cellular and molecular mechanisms.

MIP-2 causes differential activation of RhoA in mouse aortic versus pulmonary artery endothelial cells

Previously, we have shown that endothelial cell chemotaxis to the proangiogenic chemokine MIP-2 (macrophage inflammatory protein-2) is much greater in mouse aortic endothelial cells (EC) than pulmonary arterial endothelial cells (PA EC). This was true despite the observation that both cell types display comparable levels of the ligand receptor, CXCR 2 (8). Since the systemic arterial circulation is proangiogenic in the adult lung and the pulmonary circulation is relatively resistant to neovascularization, we questioned whether the observed functional heterogeneity is related to inherent differences in cell signaling cascades of the two EC subtypes. Specifically, we measured activation of Rac1 and RhoA, both thought to be involved in EC cell migration. Rac1 showed inconsistent and minimal changes in both cell types after MIP-2 treatment ( p > 0.05). However, activated RhoA was increased upon exposure to MIP-2 only in aortic EC (61% increase; p < 0.05). Decreased RhoA activation after treatment of aortic EC with specific siRNA for RhoA resulted in a functional decrease in EC chemotaxis to MIP-2 (17% increase; p < 0.05). Additionally, increased RhoA activation in PA EC with adenoviral infection of RhoA caused an increase in PA EC chemotaxis to MIP-2 (46% increase; p < 0.05). Inhibition of RhoA activity with the Rho kinase inhibitor, Y27632, blocked aortic EC chemotaxis and stress fiber formation. Thus, RhoA activation is increased after MIP-2 treatment in mouse aortic endothelial cells but not in pulmonary artery endothelial cells. We conclude that RhoA is part of a signaling pathway essential for aortic cell migration after CXCR 2 ligation. This result provides one explanation for the difference in chemotaxis observed in these two endothelial subtypes that express similar levels of CXCR 2.

Differential Effects of Phenylalanine on Rac1, Cdc42, and RhoA Expression and Activity in Cultured Cortical Neurons

Phenylketonuria (PKU) is characterized by a high concentration of phenylalanine, which can lead to mental retardation. One of the characteristic pathologic changes in untreated phenylketonuria patients is a reduction in the number of axons, dendrites, and synapses in the brain. This is thought to be due to the toxic effects of phenylalanine and/or its metabolites, however, the underlying mechanism remains unclear. In this study, we observed that phenylalanine reduced the number of dendrites and dendritic spines in cultured neurons. We further demonstrated that phenylalanine down-regulated Rac1, Cdc42, and RhoA mRNA and protein expression. Pull-down assays indicated that phenylalanine caused a decrease in Rac1/Cdc42 activity but increased RhoA activity. Expression of a dominant negative RhoA or treatment with a Rho-associated kinase specific inhibitor, Y-27632, partly inhibited the phenylalanine-induced decrease in dendrite numbers. In conclusion, we have demonstrated that phenylalanine affects the expression and activity of Rac1, Cdc42, and RhoA. Furthermore, RhoA signaling is involved in the inhibitory effect of phenylalanine on dendritic branching. These results may provide an important insight into the molecular mechanism underlying phenylalanine-induced abnormalities of dendrites, specifically in phenylketonuria neuronal injury.

Actin Cytoskeleton Dynamics Promotes Leptin-Induced Vascular Smooth Muscle Hypertrophy via RhoA/ROCK- and Phosphatidylinositol 3-Kinase/Protein Kinase B-Dependent Pathways

Obesity is associated with increased leptin production that may contribute to cardiovascular pathology through a multiplicity of effects. Leptin has been shown to contribute to vascular remodeling through various mechanisms, including production of vascular smooth muscle (VSMC) hypertrophy; however, the mechanisms underlying the vascular hypertrophic effect of leptin remain unknown. In the present study, we investigated the contributions of the RhoA/Rho kinase (ROCK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways, actin dynamics, and the expression of serum-response factor (SRF) in the hypertrophic effects of leptin on vascular tissue. Strips of rat portal vein (RPV) were cultured with or without leptin at 3.1 nM for 1 to 3 days. Leptin significantly increased RhoA activity by 163 +/- 20%, whereas phosphorylation of downstream factors, including LIM kinase 1 and cofilin-2, was increased by 160 +/- 25 and 290 +/- 25%, respectively. Leptin also significantly phosphorylated Akt by 130 +/- 30%, which was inhibited by the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). RhoA/ROCK and PI3K/Akt activation was associated with a significant increase in RPV wet weight (11 +/- 1%), protein synthesis (45 +/- 7%), SRF expression (136 +/- 11%), and polymerization of actin, as reflected by an increase in the F-/G-actin ratio, effects that were significantly attenuated by a leptin receptor (leptin obese receptor) antibody, the ROCK inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) (Y-27632) as well as the PI3K inhibitor LY294002. Our results indicate that the activation of RhoA/ROCK and PI3K/Akt plays a pivotal role in leptin signaling, leading to the development of VSMC hypertrophy through a mechanism involving altered actin dynamics.

Up-Regulation of Myometrial RHO Effector Proteins (PKN1 and DIAPH1) and CPI-17 (PPP1R14A) Phosphorylation in Human Pregnancy Is Associated with Increased GTP-RHOA in Spontaneous Preterm Labor1

RHO GTP-binding proteins are important regulators of actin-myosin interactions in uterine smooth muscle cells. Active (GTP-bound) RHOA binds to RHO-associated protein kinase (ROCK1), which inhibits the myosin-binding subunit (PPP1R12A) of myosin light chain phosphatase, leading to calcium-independent increases in myosin light chain phosphorylation and tension, which are termed "calcium sensitization." The RHO effector protein kinase N (PKN1) also increases calcium sensitization by phosphorylating the protein kinase C (PRKCB)-dependent protein CPI-17 (PPP1R14A) to inhibit the PPP1c subunit of myosin phosphatase. Moreover, other RHO proteins, such as RHOB, RHOD, and their effectors (DIAPH1 and DIAPH2), may modulate PKN1/ ROCK1 signaling to effect changes in myosin phosphatase activity and myosin light chain phosphorylation. The increases in contractile activity observed in term and preterm labor may be due to an increase in RHO activity and/or changes in RHO-related proteins. We found that the RHOA and RHOB mRNA levels in the myometrium were increased in pregnancy, although the expression levels of the RHOA and RHOB proteins did not change with pregnancy or labor. GTP-bound RHOA was increased in pregnancy, and this increase was significant in spontaneous preterm labor myometrium. PKN1 expression and PPP1R14A phosphorylation were dramatically increased in the pregnant myometrium. We also observed increases in DIAPH1 expression in spontaneous term and preterm labor myometrial tissues. The present study shows that human pregnancy is characterized by increases in PKN1 expression and PPP1R14A phosphorylation in the myometrium. Moreover, increases in GTP-bound RHOA and DIAPH1 expression may contribute to the increase in uterine activity in idiopathic preterm labor.

Nox1 Redox Signaling Mediates Oncogenic Ras-induced Disruption of Stress Fibers and Focal Adhesions by Down-regulating Rho

Generation of reactive oxygen species (ROS) by Ras oncogene-induced NADPH oxidase (Nox) 1 is required for Ras transformation phenotypes including anchorage-independent growth, morphological transformation, and tumorigenesity, but the signaling mechanism downstream of Nox1 remains elusive. Rho is known to be a critical regulator of actin stress fiber formation. Nonetheless, Rho was reported to no longer couple to loss of actin stress fibers in Ras-transformed Swiss3T3 cells despite the elevation of Rho activity. In this study, however, we demonstrate that Rho is inactivated in K-Ras-transformed normal rat kidney cells, and that abrogation of Nox1-generated ROS by Nox1 small interference RNAs or diphenyleneiodonium restores Rho activation, suggesting that Nox1-generated oxidants mediate down-regulation of the Rho activity. This down-regulation involves oxidative inactivation of the low molecular weight protein-tyrosine phosphatase by Nox1-generated ROS and a subsequent elevation in the tyrosine-phosphorylated active form of p190RhoGAP, the direct target of the phosphatase. Furthermore, the decreased Rho activity leads to disruption of both actin stress fibers and focal adhesions in Ras-transformed cells. As for Rac1, Rac1 also appears to participate in the down-regulation of Rho via Nox1. Our discovery defines a mediating role of Nox1-redox signaling for Ras oncogene-induced actin cytoskeletal changes.

Role of Rho-GTPases in complement-mediated glomerular epithelial cell injury

Visceral glomerular epithelial cells (GEC) are essential for maintenance of normal glomerular permselectivity. The actin cytoskeleton is a key determinant of GEC morphology and function. In the rat passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces nonlytic GEC injury associated with morphological changes of GEC and proteinuria. The current study addresses the role of Rho family of small GTPases in complement-mediated GEC injury. When cultured rat GEC were stimulated with complement C5b-9 for 18 h, RhoA activity increased, whereas Rac1/Cdc42 activities decreased, compared with control cells. Similar changes in Rho-GTPase activities were observed in glomeruli from rats with PHN. The amount of active p190RhoGAP, a negative upstream regulator of RhoA, was decreased in complement-stimulated GEC, potentially contributing to increased RhoA activity. To address the functional effects of Rho-GTPases, GEC were transfected with constitutively active (CA) or dominant negative (DN) Rho-GTPase mutants. GEC transfected with CA-RhoA showed a smaller and round contour and prominent cortical F-actin. In contrast, GEC transfected with CA-Rac1 demonstrated morphological changes that resembled process formation. In addition, expression of CA-RhoA attenuated complement-mediated cytotoxicity, whereas cytotoxicity was augmented by DN-RhoA. Thus exposure of GEC to complement alters the balance of RhoA, Rac1, and Cdc42 activities. The activity of Rac1 may contribute to process formation, while activation of RhoA (e.g., in the setting of complement attack), with or without blunting of Rac1 activity, may have an opposite effect, i.e., contribute to foot process effacement. Activation of RhoA increases the resistance of GEC to complement-mediated injury.

The ADMA/DDAH pathway is a critical regulator of endothelial cell motility

Asymmetric dimethylarginine (ADMA) is an inhibitor of nitric oxide production associated with abnormal blood vessel growth and repair, however, the mechanism of action of ADMA is not well understood. We studied the role of exogenous and endogenous ADMA in the regulation of cell motility and actin cytoskeleton in porcine pulmonary endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMECs) from knockout mice that lack one of the enzyme metabolising ADMA, dimethylarginine dimethylaminohydrolase I (DDAHI) as well as endothelial cells overexpressing DDAH in vitro. We show that ADMA induced stress fibre and focal adhesion formation and inhibited cell motility in primary pulmonary endothelial cells. The effects of ADMA depended on the activity of RhoA and Rho kinase and were reversed by overexpression of DDAH, nitric oxide donors and protein kinase G activator, 8-bromo-cGMP. ADMA also inhibited the activities of Rac1 and Cdc42 in cells but these changes had a minor effect on cell motility. Endogenous ADMA increased RhoA activity and inhibited cell motility in PMECs from DDAHI knockout mice and inhibited angiogenesis in vitro. These results are the first demonstration that metabolism of cardiovascular risk factor ADMA regulates endothelial cell motility, an important factor in angiogenesis and vascular repair.

Increased arginase activity and endothelial dysfunction in human inflammatory bowel disease

Nitric oxide (.NO) generation from conversion of l-arginine to citrulline by nitric oxide synthase isoforms plays a critical role in vascular homeostasis. Loss of .NO is linked to vascular pathophysiology and is decreased in chronically inflamed gut blood vessels in inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis). Mechanisms underlying decreased .NO production in IBD gut microvessels are not fully characterized. Loss of .NO generation may result from increased arginase (AR) activity, which enzymatically competes with nitric oxide synthase for the common substrate l-arginine. We characterized AR expression in IBD microvessels and endothelial cells and its contribution to decreased .NO production. AR expression was assessed in resected gut tissues and human intestinal microvascular endothelial cells (HIMEC). AR expression significantly increased in both ulcerative colitis and Crohn's disease microvessels and submucosal tissues compared with normal. TNF-alpha/lipopolysaccharide increased AR activity, mRNA and protein expression in HIMEC in a time-dependent fashion. RhoA/ROCK pathway, a negative regulator of .NO generation in endothelial cells, was examined. The RhoA inhibitor C3 exoenzyme and the ROCK inhibitor Y-27632 both attenuated TNF-alpha/lipopolysaccharide-induced MAPK activation and blocked AR expression in HIMEC. A significantly higher AR activity and increased RhoA activity were observed in IBD submucosal tissues surrounding microvessels compared with normal control gut tissue. Functionally, inhibition of AR activity decreased leukocyte binding to HIMEC in an adhesion assay. Loss of .NO production in IBD microvessels is linked to enhanced levels of AR in intestinal endothelial cells exposed to chronic inflammation in vivo.

TNF‐α‐induced activation of RhoA/Rho kinase pathway in human pulmonary microvascular endothelial cells

TNF-α induces complex signaling events in endothelial cells (ECs), resulting in gene transcription and increased junctional permeability. This study examined the activation of the RhoA/Rho kinase pathway induced by TNF-α in primary human pulmonary microvascular ECs and its role in EC responses to TNF-α. Activation of RhoA was induced by TNF-α within five minutes. This increase in RhoA activity rapidly decreased, and a late activation of RhoA was induced three hours after TNF-α treatment. This late but not early RhoA activation was associated with its accumulation in focal aggregates near EC edges. TNF-α also induced Rho kinase activation that was observed within five minutes and lasted for at least three hours. Inhibition of Rho kinase prevented TNF-α-induced cytoskeletal changes and permeability increases. TNF-α also induced activation of JNK that peaked at fifteen minutes and lasted for at least three hours, and inhibition of Rho kinase prevented TNF-α-induced early and late JNK activation. Inhibition of JNK activation, however, did not prevent TNF-α-induced cytoskeletal changes, suggesting that Rho kinase did not modulate cytoskeletal changes through JNK activation. These data demonstrate that RhoA/Rho kinase pathway is rapidly activated by TNF-α and regulates several downstream responses that include JNK activation, cytoskeletal changes and increases in permeability. Supported by NIH HL070009.

RhoA and Rho kinase activity is increased in scleroderma microvascular smooth muscle cells

Scleroderma (SSc) is a devastating illness characterized by Raynaud’s phenomenon and tissue fibrosis. However, despite the prominent role of RhoA and Rho kinase (ROCK) in mediating cold-induced vasoconstriction and myofibroblast formation, the potential dysregulation of this pathway in SSc has not been studied. The present experiments were performed to analyze RhoA/ROCK activity in SSc vascular smooth muscle cells (VSMCs). VSMCs were cultured from cutaneous arterioles, which were isolated from upper arm skin punch biopsies taken from SSc patients and age/sex-matched controls. Cells were studied at passages 9–12 after being made quiescent for 72 h. RhoA activity was assessed by pulldown assay and ROCK activity by immunoblot analysis of MYPT-1 phosphorylation. Baseline activity of RhoA was higher in SSc compared to control cells. Exposure to cold (28°C, 30 min) or serum (30 min) increased RhoA activity to a greater extent in SSc compared to control cells. Stimulation of α2-adrenoceptors (UK 14,304, 0.1 μM, 10 min) did not alter ROCK activity in control cells but significantly increased activity in SSc cells. These results indicate that the RhoA/ROCK pathway is disproportionately upregulated in SSc cells compared to control VSMCs under resting conditions and in response to cold, adrenergic, and serum stimulation. This dysregulation of the RhoA/ROCK pathway may contribute to the pathogenesis of SSc.

IpaA Targets β1 Integrins and Rho to Promote Actin Cytoskeleton Rearrangements Necessary for Shigella Entry

Shigella invasion into the colonic epithelium involves many steps including the formation of large membrane protrusions by the epithelial cells that facilitate bacterial engulfment. IpaA, a Shigella protein secreted into target cells upon cell contact induces a loss of actin stress fibers in cells and promotes the reorganization of actin at the site of entry. The mechanism for this is not known but is thought to involve recruitment of the focal adhesion protein vinculin to IpaA. Here we have examined the mechanism for the effects of IpaA on the actin cytoskeleton. We show that IpaA-induced loss of actin stress fibers and cell rounding do not require vinculin expression or an intact vinculin binding site on IpaA. Rather, we find that cells expressing IpaA exhibited elevated Rho activity and increased myosin light chain phosphorylation. In addition, IpaA decreases integrin affinity for extracellular matrix ligands by interfering with talin recruitment to the integrin cytoplasmic tail. The combination of these two effects, namely weakened adhesion and increased contractility, account for the loss of actin stress fibers and cell rounding observed in cells exposed to IpaA.

RhoA‐mediated, tumor necrosis factor α–induced activation of NF‐κB in rheumatoid synoviocytes: Inhibitory effect of simvastatin

Increasing evidence indicates that RhoA may play a central role in the inflammatory response. This study was conducted to examine the role of RhoA in mediating the activation of NF-kappaB in tumor necrosis factor alpha (TNFalpha)-stimulated rheumatoid synoviocytes, and to evaluate the modulatory effects of statins on the TNFalpha-induced activation of RhoA and NF-kappaB and the secretion of proinflammatory cytokines by rheumatoid synoviocytes. Rheumatoid synoviocytes obtained from patients with active rheumatoid arthritis were stimulated with TNFalpha and incubated with simvastatin (SMV) (1 muM). RhoA activity was assessed by a pull-down assay. NF-kappaB DNA binding activity and nuclear translocation of NF-kappaB were measured by a sensitive multiwell colorimetric assay and confocal fluorescence microscopy, respectively. TNFalpha stimulation elicited a robust increase in RhoA activity in a dose-dependent manner, and SMV mitigated this increase. TNFalpha also hastened NF-kappaB nuclear translocation of subunit p65 and increased DNA binding activity, luciferase reporter gene expression, degradation of IkappaB, and secretion of interleukin-1beta (IL-1beta) and IL-6. SMV prevented the increase in NF-kappaB activation and rise in IL-1beta and IL-6 levels induced by TNFalpha, whereas mevalonate and geranylgeranyl pyrophosphate reversed the inhibitory effects of SMV on activation of NF-kappaB and RhoA. Furthermore, cotransfection with a dominant-negative mutant of RhoA demonstrated that the TNFalpha-induced signaling pathway involved sequential activation of RhoA, leading to NF-kappaB activation and, ultimately, to secretion of cytokines. This study identifies RhoA as the key regulator of TNFalpha-induced NF-kappaB activation, which ultimately results in the secretion of proinflammatory cytokines in rheumatoid synoviocytes. The findings provide a new rationale for the antiinflammatory effects of statins in inflammatory arthritis.

The EphB4 Receptor-tyrosine Kinase Promotes the Migration of Melanoma Cells through Rho-mediated Actin Cytoskeleton Reorganization

Several studies have reported the up-regulation of EphB receptor-tyrosine kinases and ephrin-B ligands in a variety of tumors, suggesting a functional relation between EphB/ephrin-B signaling and tumor progression. The ability of the EphB receptors to regulate cell migration and promote angiogenesis likely contributes to tumor progression and metastasis. Here we show that EphB receptors, and especially EphB4, regulate the migration of murine melanoma cells. Highly malignant melanoma cells express the highest levels of EphB4 receptor and migrate faster than less malignant melanoma cells. Furthermore, inhibition of EphB receptor forward signaling by overexpression of a form of EphB4 lacking the cytoplasmic portion or by treatment with competitively acting soluble EphB2-Fc results in slower melanoma cell migration. In contrast, overexpression of active EphB4 significantly enhances cell migration. The effects of EphB4 receptor on cell migration and cell morphology require its kinase activity because the inhibition of EphB4 kinase activity by overexpression of kinase dead EphB4 inhibits cell migration and affects the organization of actin cytoskeleton. Activation of EphB4 receptor with its ligand ephrin-B2-Fc enhances the migratory ability of melanoma cells and increases RhoA activity, whereas inhibiting EphB receptor forward signaling decreases RhoA activity. Moreover, expression of dominant negative RhoA blocks the effects of active EphB4 on cell migration and actin organization. These data suggest that EphB4 forward signaling contributes to the high migratory ability of invasive melanoma cells by influencing RhoA-mediated actin cytoskeleton reorganization.

An inhibitory role for FAK in regulating proliferation: a link between limited adhesion and RhoA-ROCK signaling

Focal adhesion kinase (FAK) transduces cell adhesion to the extracellular matrix into proliferative signals. We show that FAK overexpression induced proliferation in endothelial cells, which are normally growth arrested by limited adhesion. Interestingly, displacement of FAK from adhesions by using a FAK−/− cell line or by expressing the C-terminal fragment FRNK also caused an escape of adhesion-regulated growth arrest, suggesting dual positive and negative roles for FAK in growth regulation. Expressing kinase-dead FAK-Y397F in FAK−/− cells prevented uncontrolled growth, demonstrating the antiproliferative function of inactive FAK. Unlike FAK overexpression–induced growth, loss of growth control in FAK−/− or FRNK-expressing cells increased RhoA activity, cytoskeletal tension, and focal adhesion formation. ROCK inhibition rescued adhesion-dependent growth control in these cells, and expression of constitutively active RhoA or ROCK dysregulated growth. These findings demonstrate the ability of FAK to suppress and promote growth, and underscore the importance of multiple mechanisms, even from one molecule, to control cell proliferation.

RRIG1 Mediates Effects of Retinoic Acid Receptor β2 on Tumor Cell Growth and Gene Expression through Binding to and Inhibition of RhoA

The expression of retinoic acid receptor beta2 (RAR-beta2) is frequently lost in various cancers and their premalignant lesions. However, the restoration of RAR-beta2 expression inhibits tumor cell growth and suppresses cancer development. To understand the molecular mechanisms responsible for this RAR-beta2-mediated antitumor activity, we did restriction fragment differential display-PCR and cloned a novel retinoid receptor-induced gene 1 (RRIG1), which is differentially expressed in RAR-beta2-positive and RAR-beta2-negative tumor cells. RRIG1 cDNA contains 2,851 bp and encodes a protein with 276 amino acids; the gene is localized at chromosome 9q34. Expressed in a broad range of normal tissues, RRIG1 is also lost in various cancer specimens. RRIG1 mediates the effect of RAR-beta2 on cell growth and gene expression (e.g., extracellular signal-regulated kinase 1/2 and cyclooxygenase-2). The RRIG1 protein is expressed in the cell membrane and binds to and inhibits the activity of a small GTPase RhoA. Whereas induction of RRIG1 expression inhibits RhoA activation and f-actin formation and consequently reduces colony formation, invasion, and proliferation of esophageal cancer cells, antisense RRIG1 increases RhoA activity and f-actin formation and thus induces the colony formation, invasion, and proliferation of these cells. Our findings therefore show a novel molecular pathway involving RAR-beta2 regulation of RRIG1 expression and RRIG1-RhoA interaction. An understanding of this pathway may translate into better control of human cancer.

P120 Catenin Regulates Dendritic Spine and Synapse Development through Rho-Family GTPases and Cadherins

Both the cadherin-catenin complex and Rho-family GTPases have been shown to regulate dendrite development. We show here a role for p120 catenin (p120ctn) in regulating spine and synapse formation in the developing mouse brain. p120catenin gene deletion in hippocampal pyramidal neurons in vivo resulted in reduced spine and synapse densities along dendrites. In addition, p120 catenin loss resulted in reduced cadherin levels and misregulation of Rho-family GTPases, with decreased Rac1 and increased RhoA activity. Analyses in vitro indicate that the reduced spine density reflects aberrant Rho-family GTPase signaling, whereas the effects on spine maturation appear to result from reduced cadherin levels and possibly aberrant Rho-family GTPase signaling. Thus, p120ctn acts as a signal coordinator between cadherins and Rho-family GTPases to regulate cytoskeletal changes required during spine and synapse development.

Cingulin Regulates Claudin-2 Expression and Cell Proliferation through the Small GTPase RhoA

In mouse embryoid bodies, mutation of the tight junction protein cingulin results in changes in gene expression. Here, we studied the function of cingulin using a gene silencing approach in Madin-Darby canine kidney (MDCK) cells. Cingulin-depleted cells show higher protein and mRNA levels of claudin-2 and ZO-3, increased RhoA activity, activation of G1/S phase transition, and increased cell density. The effects of cingulin depletion on claudin-2 expression, cell proliferation, and density are reversed by coexpression of either a dominant-negative form of RhoA (RhoAN19) or the Rho-inhibiting enzyme C3 transferase. However, the increase in ZO-3 protein and mRNA levels is not reversed by inhibition of either RhoA, p38, extracellular signal-regulated kinase (ERK), or c-Jun NH2-terminal kinase (JNK), suggesting that cingulin modulates ZO-3 expression by a different mechanism. JNK is implicated in the regulation of claudin-2 levels independently of cingulin depletion and RhoA activity, indicating distinct roles of RhoA- and JNK-dependent pathways in the control of claudin-2 expression. Finally, cingulin depletion does not significantly alter the barrier function of monolayers and the overall molecular organization of tight junctions. These results provide novel insights about the mechanisms of cingulin function and the signaling pathways controlling claudin-2 expression in MDCK cells.

RhoA and p38 MAPK mediate apoptosis induced by cellular cholesterol depletion

Cholesterol is essential for cell viability, and homeostasis of cellular cholesterol is crucial to various cell functions. Here we examined the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in NIH3T3 cells. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment resulted in a significant increase in cellular apoptosis and caspase-3 activation. This effect is not due to a deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, but rather to low cholesterol levels, since addition of cholesterol together with LPDS and 25-HC nearly abolished apoptosis, whereas addition of farnesyl pyrophosphate or geranylgeranyl-pyrophosphate did not reverse the cell viability loss induced by LPDS plus 25-HC treatment. These effects were accompanied by an increase in ERK, JNK and p38 MAPK activity. However, only the inhibition of p38 MAPK with the specific inhibitor SB203580 or the overexpression of a kinase defective MKK6 resulted in a significant decrease in apoptosis and caspase-3 cleavage induced by cholesterol depletion. Furthermore, LPDS plus 25-HC increased RhoA activity, and this effect was reversed by addition of exogenous cholesterol. Finally, overexpression of the dominant negative N19RhoA inhibited p38 MAPK phosphorylation and apoptosis induced by low cholesterol levels. Together, our results demonstrate that cholesterol depletion induces apoptosis through a RhoA- and p38 MAPK-dependent mechanism.