RHOG Activates RAC1 Through CDC42 Leading to Tube Formation in Vascular Endothelial Cells

cells

Article RHOG Activates RAC1 through CDC42 Leading to Tube Formation in Vascular Endothelial Cells

Oula El Atat, Amira Fakih and Mirvat El-Sibai * Department of Natural Sciences, Lebanese American University, Beirut 1102 2801, Lebanon; [email protected] (O.E.A.); [email protected] (A.F.) * Correspondence: [email protected]; Tel.: +961-1-786456 (ext. 1253); Fax: +961-1-8670984

Received: 9 January 2019; Accepted: 13 February 2019; Published: 18 February 2019

Abstract: Angiogenesis is a hallmark of cancer cell malignancy. The role of the RHO family GTPase RHOG in angiogenesis in vascular endothelial cells has recently been elucidated. However, the regulation of RHOG during this process, as well as its cross-talk with other RHO GTPases, have yet to be fully examined. In this study, we found that siRNA-mediated depletion of RHOG strongly inhibits tube formation in vascular endothelial cells (ECV cells), an effect reversed by transfecting dominant active constructs of CDC42 or RAC1 in the RHOG-depleted cells. We also found CDC42 to be upstream from RAC1 in these cells. Inhibiting either Phosphatidyl inositol (3) kinase (PI3K) with Wortmannin or the mitogen-activated protein kinase extracellular-regulated kinase (MAPK ERK) with U0126 leads to the inhibition of tube formation. While knocking down either RHO, GTPase did not affect p-AKT levels, and p-ERK decreased in response to the knocking down of RHOG, CDC42 or RAC1. Recovering active RHO GTPases in U0126-treated cells also did not reverse the inhibition of tube formation, placing ERK downstream from PI3K-RHOG-CDC42-RAC1 in vascular endothelial cells. Finally, RHOA and the Rho activated protein kinases ROCK1 and ROCK2 positively regulated tube formation independently of ERK, while RHOC seemed to inhibit the process. Collectively, our data conﬁrmed the essential role of RHOG in angiogenesis, shedding light on a potential new therapeutic target for cancer malignancy and metastasis.

Keywords: RHOG; RAC1; CDC42; RHO GTPases; angiogenesis; vascular endothelial cells

1. Introduction Angiogenesis is the formation of new blood vessels from preexisting blood vessels [1]. It is required during embryonic development, growth, regeneration and wound healing [1,2]. Furthermore, it is implicated in pathological processes including arthritis, muscular dystrophy, and tumorigenesis [3]. During tumorigenesis, the angiogenic process starts when endothelial cells (EC) branch from pre-existing small vessels, forming sprouts of capillaries that can supply the tissue of the tumor [2]. The new blood vessel formation is triggered by angiogenic stimuli, such as the vascular endothelial growth factor (VEGF) [1,4]. Growth factors stimulate the proliferation and migration of ECs through the activation of the phosphatidylinositol (3) kinase (PI3K) pathway as well as the Mitogen-activated protein kinase (MAPK) pathway [5–7]. New blood vessel formation is also regulated by the RHO GTPase family [8–10]. The RHO family of GTPases is comprised of 20 members of small GTP binding proteins with molecular sizes ranging from 20–40 KDa [11]. The most considered RHO GTPases are RHOA/C, RAC1, and CDC42 [12–14]. RHO GTPases act as switches between active GTP-binding states and inactive GDP-binding states [11]. They are activated by the guanine nucleotide exchange factors, (GEFs) which are, in many cell types, activated downstream from PI3K [15,16]. GTPases-activating proteins (GAPs) and Guanine dissociation inhibitors (GDIs) lead to the inactivation of RHO GTPases (Schmidt & Hall,

Cells 2019, 8, 171; doi:10.3390/cells8020171 www.mdpi.com/journal/cells Cells 2019, 8, 171 2 of 17

2002). RHO GTPases are key regulators of the actin cytoskeleton and actin remodeling events required during cellular proliferation and migration [17–19]. RHOA, RHOC, RAC1, and CDC42 regulate endothelial cell proliferation, polarization, cell-cell adhesion, and migration, as well as vascular permeability during angiogenesis [8,9,20–25]. RHOG is an emerging RHO GTPase that possesses almost the same sequence as RAC1 and CDC42 and that has been found to be critical for the regulation of the actin cytoskeleton and migration in many cell types [26–32]. In addition, several reports also established a cross-talk between RHOG and RAC1 and CDC42, where RHOG was found to activate CDC42/RAC1 in some systems [28,31,33,34]. To date, the role of RHOG in vascular endothelial cells during angiogenesis has not been elucidated. In this study, we investigated the role of RHOG in tube formation in vascular endothelial cells. We also examined its regulation, cross-talk with other RHO GTPases, and potential downstream effectors. This established RHOG as a potentially promising new target for anti-angiogenesis therapeutic applications in cancer.

2. Materials and Methods

2.1. Cells and Culture Conditions Human endothelial cells ECV were cultured in RPMI 1640 supplemented with 10% Fetal Bovine ◦ Serum (FBS) and 100 U/µL of penicillin/streptomycin at 37 C and 5% CO2 in a humidiﬁed chamber.

2.2. Antibodies and Reagents The following primary antibodies were used in this study: Mouse monoclonal anti-RHOG, mouse monoclonal anti-RHOA, rabbit polyclonal anti-RHOC, mouse monoclonal anti-RAC1, rabbit monoclonal anti-ROCK1, rabbit monoclonal anti-ROCK2, rabbit polyclonal anti-p-AKT1 (phospho-S473), rabbit polyclonal anti-pan-AKT, and rabbit polyclonal anti-Actin antibodies, as well as the MEK1/2 inhibitor U0126 and the PI3K inhibitor Wortmannin, which were obtained from Abcam (Abcam Inc., Cambridge, UK). Rabbit polyclonal anti-CDC42 (sc-87), mouse monoclonal anti-p-ERK1 (phospho-Y204), mouse monoclonal anti-ERK, and goat polyclonal anti-STARD13 antibodies were obtained from Santa Cruz (Santa Cruz Biotechnology., Santa Cruz, CA, US). Anti-rabbit and anti-mouse HRP-conjugated secondary antibodies were obtained from Promega (Promega Co., Maddison, WI, USA). The Dominant active Cherry-tagged RAC1-Q61L and Cherry-tagged CDC42-Q61L constructs were a generous gift from Dr. Louis Hodgson.

2.3. Cell Transfection and Small Interfering RNA Cells were transfected with 5 µg dominant active RAC1, dominant active CDC42, or the control empty vector (pcDNA3.1) using Lipfectamine LTX with Plus reagent (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer. The experiments were performed 24 h following transfection. Human FlexiTube siRNA for each of RHOG [oligo 1, 2 and 3 out of the NM_001665 panel), RAC1 (oligo 5 and 6 out of the NM_006908, NM_018890 and NM_198829 panels), CDC42 (oligo 4 and 7 out of the NM_001039802, NM_001791 and NM_044472 panels), STARD13 (oligo 4 out of the NM_001243466 panel], RHOA (oligo 1 and 6 out of the NM_001664 panel), RHOC (oligo 5 and 6 out of the NM_001042678, NM_00104269 and NM_175744 panels), ROCK1 (oligo 9 and 10 out of the NM_005406 panel) and ROCK2 (oligo 5 and 6 out of the NM_004850 panel) were brought from Qiagen (Qiagen, Hilden, Germany). The cells were transfected with the siRNA at a ﬁnal concentration of 10 nM using HiPerfect (Qiagen, Hilden, Germany) as described by the manufacturer. Control cells were transfected with siRNA sequences targeting GL2 Luciferase (Qiagen, Hilden, Germany). After 72 h, the protein levels in the total cell lysates were analyzed by Western blotting using the appropriate antibodies, or the effect of the corresponding knockdown was assayed. Western blot analyses were performed to choose the oligos showing the most effective knockdowns from every FlexiTube panel corresponding to the target sequence of proteins mentioned above. These oligos were used in this study, and Western blots Cells 2019, 8, 171 3 of 17 showing knockdown efﬁciency were also included in the study. Cells transfected with both siRNA and a vector were incubated with the siRNA for 72 h as previously mentioned then co-transfected with the appropriate vector 24 h prior to the experiment.

2.4. Immunoblotting Cell lysates were prepared by scraping the cells with Laemmli Sample Buffer (LSB) containing 4% Sodium Dodecyl Sulfate SDS, 20% Glycerol, 10% β-Mercaptoethanol, 0.004% Bromophenol Blue, and 0.125 M Tris-HCl (pH = 6.8). The resulting lysates were boiled for 5 min. Protein samples were separated by SDS-PAGE gels and transferred to PVDF membranes. Membranes were then blocked either by 5% BSA for 2 h at room temperature depending on the primary antibody used. The membranes were then incubated with the appropriate primary antibody for 2 h. After that, the membranes were washed and incubated with secondary antibodies at a concentration of 1:2000 for 1 h at room temperature. Bands were visualized by treating the membranes with a chemiluminescent reagent Enhanced Chemiluminescence-ECL detection kit (GE Healthcare, Chicago, IL, USA). The levels of expression of proteins were compared by densitometry using the ImageJ software and plotted in Excel.

2.5. Pull Down Assays The pull-down assays were performed using the RHOA/RAC1/CDC42 Activation Assay Combo Kit (Cell BioLabs, San Diego, CA, USA) following the manufacturer’s instructions. Brieﬂy, cell lysates were incubated with GST-RBD (for RHOA pull-down) or GST-CRIB (for RAC1/CDC42 pull-down) for 1 h at 4 ◦C with gentle agitation. Then, the samples were centrifuged, and the pellet was washed several times. After the last wash, the pellets were resuspended with sample buffer and boiled for 5 min. GTP-RHOA, GTP-RAC1, and GTP-CDC42 were detected by Western blotting using anti-RHOA, anti-RAC1, and anti-CDC42, respectively. Total RHOA/RAC1 and CDC42 were collected prior to the incubation with GST-RBD/GST-CRIB and used as a loading control. All Western blots were analyzed with ECL (GE Healthcare, Chicago, Il, USA) followed by densitometry.

2.6. The Tube Formation Assay Matrigel™ basement membrane matrix (growth factor reduced) (BD Biosciences, San Jose, CA, USA) was thawed at 4 ◦C, pipetted into pre-cooled 35 mm Petri dishes, and incubated at 37 ◦C for 1 h. After Matrigel polymeration, treated cells were suspended in RPMI medium and were seeded onto the Matrigel. For tube formation analysis, images were taken using inﬁnity corrected optics on a Nikon Eclipse microscope supplemented with a computer-driven Roper cooled CCD camera and operated by IPLab Spectrum software v. 2.8 (BD Bioscience, San Jose, CA, USA, 2012) after 24, 48, and 72 h using 10× objective. The tube formation assay was analyzed and interpreted using the WimTube software (Wimasis, Munich, Germany, 2015, Image Analysis,) based on different parameters such as branching points, tube lengths, tube numbers, loop numbers, areas, and perimeters, and cell covered area.

2.7. Statistical Analysis All the results reported represent average values from three independent experiments. All error estimates are given as the mean ± standard error of the mean (S.E.M). Statistical differences were determined using one-way analysis of variance. The p-values of less than 0.05 were considered significant.

3. Results

3.1. RHOG Positively Regulates Tube Formation in ECV Cells To investigate the role of RHOG in angiogenesis, RHOG was knocked down using small interfering siRNA in ECV cells and a tube formation assay was performed. The efﬁciency of the knockdown was determined using Western blot (Figure1A). The 3 different siRNA used reduced CellsCells2019 2019, 8, 171, 8, x 4 4of of 18 17

RHOG expression by 60–80% in the transfected cells compared to Luciferase siRNA-transfected RHOG expression by 60–80% in the transfected cells compared to Luciferase siRNA-transfected control control cells (β−actin was used as loading control) (Figure 1A,B). Then, the effect of RHOG β− cellsknockdown ( actin wason angiogenesis used as loading was determined control) (Figure by testing1A,B). the Then, effect the of effectRHOG of knockdown RHOG knockdown in a tube on angiogenesisformation assay. was The determined Wimtube Softwa by testingre was the used effect to analyze of RHOG the total knockdown tube length, in athe tube total formation number assay.of tubes, The Wimtube and branching Software points was before used and to analyze after the the knockdowns. total tube length, Our results the total showed number a significant of tubes, anddecrease branching (approximately points before 50%) and in after the total the knockdowns.tube length in response Our results to the showed RHOG aknockdown signiﬁcant at decrease 24, 48, (approximatelyand 72 h post-plating 50%) in the (Figure total tube1C,D). length The number in response of tubes to the in RHOGaddition knockdown to the number at 24, of 48, branching and 72 h post-platingpoints at (Figureeach of1 theC,D). three The time number intervals of tubes also insh additionowed a decrease to the number (Figure of 1C,E,F). branching This points showed at that each of thethe three formation time intervalsof blood vessels also showed in vascular a decrease endothelia (Figurel cells,1C,E,F). as reflected This showed in the tube that formation the formation assay, of bloodis RHOG-dependent. vessels in vascular endothelial cells, as reﬂected in the tube formation assay, is RHOG-dependent.

FigureFigure 1. RHOG 1. RHOG positively positively regulates regulates tube tube formation formation in in ECV ECVcells. cells. ECVECV cells were transfected with with luciferaseluciferase control control siRNA siRNA or or with with RHOG RHOG siRNA. siRNA. Three Three different different siRNAsiRNA oligosoligos against RHOG RHOG were were usedused in eachin each experiment. experiment. (A (A)) The The cells cells werewere lysed lysed and and immunoblo immunoblottedtted using using Western Western blot blot analysis analysis for for RHOG (upper gel) or for actin (lower gel) for the loading control. (B (B) )Western Western blot blot bands bands were were quantifiedquantified using using imageJ imageJ and normalizedand normalized to the to number the nu ofmber total of proteins total proteins and expressed and expressed as fold decreases as fold fromdecreases the luciferase from the control. luciferase Data control. are the Data mean are ±theSEM mean of ± three SEM independentof three independent experiments. experiments. * p < 0.05* p < 0.05 indicates statistically significant differences. (C) Representative images of the tube formation indicates statistically significant differences. (C) Representative images of the tube formation assay assay on the growth factor-reduced Matrigel by ECV at 24, 48, and 72 h after plating. (D–F) on the growth factor-reduced Matrigel by ECV at 24, 48, and 72 h after plating. (D–F) Quantitation Quantitation of (C) for the total tube length, total tube number, and the number of branching points, of (C) for the total tube length, total tube number, and the number of branching points, respectively. respectively. Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates statistically significant statistically significant differences with the luciferase control. The scale bar is 100 µm. differences with the luciferase control. The scale bar is 100 µm. Cells 2019, 8, 171 5 of 17

3.2. RAC1 Positively Regulates Tube Formation in ECV Cells Since RHOGCells 2019, 8 has, x been found in many systems to be an upstream regulator of RAC15 of [1833 ], it was interesting3.2. to RAC1 examine Positively if RAC1Regulates also Tube regulatesFormation in tubeECV Cells formation in ECV cells. RAC1 was knocked down using 2 different siRNA oligos. The Western blot confirmed that RAC1 targeting siRNA Since RHOG has been found in many systems to be an upstream regulator of RAC1 [33], it was significantlyinteresting reduced to examine the protein if RAC1 levels also regulates of RAC1 tube formation (Figure2 inA,B). ECV cells. As expected,RAC1 was knocked RAC1 down knockdown resulted inusing a significant 2 different siRNA decrease oligos. in The the Western total blot tube confirmed length that and RAC1 the targeting total number siRNA significantly of tubes at 24, 48, and 72 h (Figurereduced2 C–E).the protein Moreover, levels of the RAC1 number (Figure of 2A,B branching). As expected, points RAC1 also knockdown decreased resulted upon in knockdown a significant decrease in the total tube length and the total number of tubes at 24, 48, and 72 h (Figure due to the decrease in the number of tube formations (Figure2C,F). In order to determine if RHOG 2C–E). Moreover, the number of branching points also decreased upon knockdown due to the directly regulatesdecrease RAC1in the number in these of tube cells, formations RHOG (Figur was knockede 2C,F). In down,order to anddetermine RAC1 if RHOG activation directly was tested using a pull-downregulates RAC1 assay. in these In brief, cells, RHOG cells were was knocke lysedd anddown, incubated and RAC1 activation with GST-CRIB was tested (Cdc42 using a and Rac interactivepull-down binding assay. domain In brief, from cells PAK1)were lysed for and 30 incubated min at with 4 ◦C. GST-CRIB Active (Cdc42 RAC1 and was Rac interactive then detected by binding domain from PAK1) for 30 min at 4 °C. Active RAC1 was then detected by Western blot. Western blot. Indeed, in cells transfected with RHOG siRNA, the level of active RAC1 substantially Indeed, in cells transfected with RHOG siRNA, the level of active RAC1 substantially decreased decreased (Figure 3A,B).3A,B). Furthermore, Furthermore, RHOG RHOG siRNA-transfec siRNA-transfectedted ECV cells were ECV able cells to reverse wereable the RHOG to reverse the RHOG siRNA-mediatedsiRNA-mediated tubetube formation inhibition inhibition when when co-transfected co-transfected with a withdominant a dominant active RAC1 active RAC1 construct (RAC1-Q61L)construct (RAC1-Q61L) (Figure (Figure3C,D). 3C,D).

Figure 2. RAC1Figure 2. positively RAC1 positively regulates regulates tube tube formation formation inin ECV ECV cells. cells. ECV cells ECV were cells transfected were transfected with the with luciferase control siRNA or with RAC1 siRNA. Two different siRNA oligos against RAC1 were used the luciferasein each control experiment. siRNA (A or) The with cells RAC1 were lysed siRNA. and immunoblotted Two different using siRNA Western oligos blot against analysis RAC1for were used in eachRAC1 experiment. (upper gel) (orA )for The actin cells (lower were gel) lysed for the and loading immunoblotted control. (B) Western using blot Western bands were blot analysis for RAC1 (upperquantified gel) using or imageJ for actin and (lowernormalized gel) to for the the number loading of total control. proteins ( Band) Western expressed blot as fold bands were quantified usingdecreases imageJ from the and luciferase normalized control. to Data the are number the mean of ± total SEM proteinsof three independent and expressed experiments. as fold * p decreases from the luciferase control. Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates statistically significant differences. (C) Representative images of the tube formation assay on the growth factor-reduced Matrigel by ECV after 24, 48, and 72 h after plating. (D–F) Quantitation of (C) for the total tube length, total tube number, and the number of branching points, respectively. Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates statistically significant differences with the luciferase control. The scale bar is 100 µm. Cells 2019, 8, x 6 of 18

< 0.05 indicates statistically significant differences. (C) Representative images of the tube formation assay on the growth factor-reduced Matrigel by ECV after 24, 48, and 72 h after plating. (D–F) Quantitation of (C) for the total tube length, total tube number, and the number of branching points, Cells 2019, 8, 171 respectively. Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates 6 of 17 statistically significant differences with the luciferase control. The scale bar is 100 µm.

Figure 3. RHOGFigure 3. activatesRHOG activates RAC1 RAC1 leading leading to tubeto tube formation formation in in ECV ECV cells. cells. (A) (CellsA) Cells were weretransfected transfected with eitherwith luciferase either luciferase or RHOG or RHOG siRNA. siRNA. Cells Cells were were then then lysedlysed and and incubate incubatedd with with GST-CRIB GST-CRIB (CDC42 (CDC42 and RACand interactive RAC interactive binding binding domain) domain) to to pull pull down down the the active active RAC1. RAC1. Samples Samples from the from pull-down the pull-down as well as the total lysates were blotted against RAC1. The lower 2 gels are Western blots for RHOG for as well as the total lysates were blotted against RAC1. The lower 2 gels are Western blots for RHOG the knockdown control and actin for the loading control. (B) Quantitation of GTP-RAC1 from (A) for the knockdownnormalized controlto total RAC1 and actinand expressed for the loadingas a fold de control.crease from (B) Quantitationthe luciferase control. of GTP-RAC1 Data are the from (A) normalizedmean to total± SEM RAC1of three andindependent expressed experiments. as a fold * p decrease < 0.05 indicates from statistically the luciferase significant control. differences. Data are the mean ± SEM(C) Representative of three independent images of the experiments. tube formation * p assay< 0.05 (72 indicatesh) of ECV cells statistically treated with signiﬁcant RHOG siRNA differences. (C) Representative(left) or RHOG images siRNA/RAC1-DA of the tube (rig formationht). The scale assay bar (72 is 100 h) µm. of ECV (D) Quantitation cells treated of (C) with for RHOGthe total siRNA (left) or RHOGtube length siRNA/RAC1-DA with the luciferase (right). siRNA/vector The scale alone bar as a is control. 100 µ m.The (dataD) Quantitationare the mean ± SEM of (C) of forthree the total independent experiments. * p < 0.05 indicates statistically significant differences. tube length with the luciferase siRNA/vector alone as a control. The data are the mean ± SEM of three independent3.3. RHOG experiments. Activates RAC1 * p

which led to an approximately 80% decrease in CDC42 activation as compared to the luciferase- transfected cells (Figure 4E). In addition, when ECV cells were co-transfected with RHOG siRNA and a dominant active CDC42 construct (CDC42-Q61L), this recovered the RHOG knockdown-mediated decrease in RAC1 activation seen in cells transfected with RHOG siRNA alone (Figure 4F). This data showed CDC42/RAC1 as a signaling module downstream from RHOG in EVC cells, with CDC42 activity seeming necessary and sufficient in mediating the RHOG activation of RAC1. We also looked at a potential activation of RHOA downstream from RHOG. When either RHOG or CDC42 were Cells 2019, 8, 171 7 of 17 knocked down, the level of activation of RHOA was not affected in these cells (Supplemental Figure S1), suggesting RhoA operates in a separate pathway.

Figure 4. RHOG activates CDC42 which activates RAC1 in ECV cells. (A) Cells were transfected with Figure 4. RHOG activates CDC42 which activates RAC1 in ECV cells. (A) Cells were transfected with either theeither luciferase the luciferase control control or CDC42 or CDC42 siRNA siRNA (2 (2 oligos). oligos). The The cells cells were were then then lysed, lysed, and the and samples the samples blotted withblotted anti-CDC42 with anti-CDC42 antibody. antibody. (B) Quantitation(B) Quantitation of of (A) (knockdown (knockdown efficiency) efficiency) normalized normalized to actin to actin and expressedand expressed as a fold as a decreasefold decrease compared compared to the the lu luciferaseciferase control. control. Data are Data the mean are ± the SEMmean of three± SEM of three independentindependent experiments. experiments. * p << 0.05 indicates statistica statisticallylly significant significant differences. differences. (C) Cells (C were) Cells were transfected with either luciferase or CDC42 siRNA (as in A), lysed, and incubated with GST-CRIB to transfected with either luciferase or CDC42 siRNA (as in A), lysed, and incubated with GST-CRIB to pull down active RAC1. Samples, as well as total lysates (lower gel), were then blotted with RAC1 pull downantibody. active RAC1.The graph Samples, is a quantitation as well of asthe totalgel to lysatesthe total RAC1 (lower and gel), is expressed were then as a blottedfold decrease with RAC1 antibody.compared The graph to the is aluciferase quantitation control. of The the data gel are to the the mean total ± SEM RAC1 of three and independent is expressed experiments. as a fold * decrease comparedp to< 0.05 the indicates luciferase statistically control. significant The data differences. are themean (D) Cells± wereSEM transfected of three independentwith either luciferase experiments. * p < 0.05 indicatesor RAC1 siRNA statistically lysed and significant incubated with differences. GST-CRIB (toD pull) Cells down were active transfected CDC42. Samples, with as either well as luciferase total lysates (lower gel), were then blotted with the CDC42 antibody. The graph is a quantitation of or RAC1 siRNA lysed and incubated with GST-CRIB to pull down active CDC42. Samples, as well as the gel to total CDC42 and expressed as a fold decrease compared to the luciferase control. Data are total lysatesthe (lower mean ± gel), SEM were of three then independent blotted with experiments. the CDC42 * p antibody. < 0.05 indicates The graph statistically is a quantitation significant of the gel to totaldifferences. CDC42 and(E) Cells expressed were transfected as a fold with decrease either luci comparedferase or RHOG to the siRNA luciferase lysed control.and incubated Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates statistically significant differences. (E) Cells were transfected with either luciferase or RHOG siRNA lysed and incubated with GST-CRIB to pull down active CDC42. Samples were then blotted with anti-CDC42 and total lysates with anti-CDC42, anti-RHOG (for knockdown efficiency), and anti-actin (for loading control). The graph is a quantitation of GTP-CDC42 ratioed to total CDC42 and expressed as fold decrease compared to luciferase control. Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates statistically significant differences. (F) Cells were transfected with either luciferase control and vector alone or with RHOG siRNA and CDC42-DA. Then, cells were lysed and incubated with GST-CRIB (CDC42 and RAC1 interactive binding domain) (GTP-RAC1 and GTP-CDC42 gels) to pull down active CDC42 and RAC1. Samples were then blotted with RAC1 and CDC42 antibodies. Total cell lysates were also blotted with RAC1, CDC42 antibodies. The lower 2 gels are Western blots for RHOG and actin for knockdown efficiency.

3.4. RHOG Stimulates Tube Formation through CDC42 and RAC1 To directly confirm that CDC42 is required for tube formation, CDC42 was knocked down using the oligos described in Figure4. As expected, the CDC42 knockdown inhibited tube formation relative Cells 2019, 8, 171 8 of 17 to non-treated cells (Figure5A). Quantitative analysis confirmed that the length and numbers of the tubes, as well as branching point numbers, decreased in response to the CDC42 knockdown (Figure5B–D). We then looked at the effect of knocking down the CDC42/RHOA-GAP STARD13 on tube formation in these cells. The efficiency of the knockdown in these cells was tested by Western blot (Figure5E,F). When STARD13 was knocked down, this led to an increase in CDC42 activation, as expected (Figure5E,G). Surprisingly, STARD13 does not seem to affect RhoA activation in these cells (Supplemental Figure S1). STARD13 knockdown led to an increase in tube formation and in the totals of the length and numbers of the tubes, as well as in the branching point numbers (Figure5H–K). Finally, when dominant active CDC42 (CDC42-Q61L) or STARD13 siRNA were added to RHOG siRNA, this rescued the tube inhibition (Figure5L,M). These results confirmed that RHOG regulates tube formationCells 2019 through, 8, x the CDC42/RAC1 pathway 9 of 18

Figure 5. RHOGFigure 5. stimulates RHOG stimulates tube tube formation formation through through CDC42 and and RAC1. RAC1. ECV-cells ECV-cells were transfected were transfected with the luciferasewith the luciferase control, control, CDC42 CDC42 (2 oligos) (2 oligos) or or STARD13 STARD13 siRNA siRNA and and plated plated on growth on growth factor-reduced factor-reduced Matrigel by ECV for 24, 48, or 72 h. (A) Representative images of tube formation assay for the Matrigel by ECV for 24, 48, or 72 h. (A) Representative images of tube formation assay for the luciferase luciferase control and the CDC42 knockdown cells at 24, 48, and 72 h after plating. The scale bar is 100 µm. (B–D) Quantitation of (A) for the total tube length, total tube number, and the number of branching points, respectively. Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates statistically significant differences with the luciferase control. (E) Cells were transfected with either luciferase or STARD13 siRNA, and cells were then lysed and incubated with GST-CRIB to pull down active CDC42. The samples were then blotted with CDC42 antibody. Total cell lysates were also Cells 2019, 8, 171 9 of 17

control and the CDC42 knockdown cells at 24, 48, and 72 h after plating. The scale bar is 100 µm. (B–D) Quantitation of (A) for the total tube length, total tube number, and the number of branching points, respectively. Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates statistically significant differences with the luciferase control. (E) Cells were transfected with either luciferase or STARD13 siRNA, and cells were then lysed and incubated with GST-CRIB to pull down active CDC42. The samples were then blotted with CDC42 antibody. Total cell lysates were also blotted with STARD13 for knockdown efficiency and actin for the loading control. (F) Quantitation of STARD13 expression (cells −/+ STARD13 siRNA) in the Western blot bands from (E) normalized to actin (lowest gel) and expressed as a fold change. Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates statistically significant differences. (G) Quantitation of GTP-CDC42 from (E) normalized to total CDC42 and expressed as a fold change from the luciferase control. Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates statistically significant differences. (H) Representative images of the tube formation assay for the luciferase control and the STARD13 knockdown cells at 24, 48, and 72 h after plating. The scale bar is 100 µm. (I–K) Quantitation of (H) for the total tube length, total tube number, and the number of branching points, respectively. Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates statistically significant differences with the luciferase control. (L) Representative images of tube formation assay (72 h) of ECV cells treated with RHOG siRNA (left), RHOG/STARD13 siRNA (double knockdown) (middle), or RHOG siRNA/CDC42-DA (left). The scale bar is 100 µm. (M) Quantitation of (L) for total tube length with the luciferase siRNA/vector alone as the control. Data are the mean ± SEM of three independent experiments. * p < 0.05 indicates statistically significant differences.

3.5. RHOG/CDC42/RAC1 Leads to Tube Formation through the ERK Pathway The role of MAPK pathways (particularly the MEK/ERK pathway) in the regulation of vascular development and angiogenesis has been established [35]. Accordingly, the effect of ERK inhibition on tube formation in these cells was examined. For this treatment, the cells were plated for 48 h then treated with 10 µM of the speciﬁc MEK1/2 inhibitor U0126 for 24 h and then imaged. The U0126-treated cells showed inhibition of tube formation (Figure6A). Surprisingly, when the U0126-treated ECV cells were transfected with CDC42-DA or RAC1-DA, the cells were not able to rescue the U0126-dependent inhibition of tube formation compared to control cells (Figure6A). In addition, Western blot analysis showed a decrease in p-ERK when the cells were treated with U0126 or with RHOG, RAC1, or CDC42 siRNA (Figure6B,C). The reverse experiment was also performed. When ECV cells were treated with U0126, this did not affect CDC42 or RAC1 activation (Figure5D,E). This, collectively, suggested that RHO GTPases act upstream from the MAPK pathway, leading to tube formation in these cells. Cells 2019, 8, 171 10 of 17 Cells 2019, 8, x 11 of 18

FigureFigure 6. RHOG/CDC42/RAC1 6. RHOG/CDC42/RAC1 leadsleads to to tube tube formation formation through through ERK ERK and anddownstream downstream from PI3K. from (A PI3K.) (A) RepresentativeRepresentative images of of a a tube tube formation formation assay assay (72 (72h after h after plating) plating) of ECV of ECVcells transfected cells transfected with the with the vectorvector alone, alone, CDC42-DA,CDC42-DA, or RAC1-DA, and and treate treatedd with with DMSO DMSO or orwith with 10 10µMµ MU0126 U0126 (24 (24h before h before imaging).imaging). The The scale scale bar bar is 100is 100µm. µm. (B (,BC,C) ECV) ECV cells cells werewere transfectedtransfected either either with with luciferase, luciferase, RHOG, RHOG, CDC42, or RAC1 siRNA, or transfected with the vector alone, CDC42-DA, or RAC1-DA, and treated CDC42, or RAC1 siRNA, or transfected with the vector alone, CDC42-DA, or RAC1-DA, and treated with 10 µM U0126 (24 h) or with the carrier (DMSO). (B) Western blot of the lysates from the different with 10 µM U0126 (24 h) or with the carrier (DMSO). (B) Western blot of the lysates from the different conditions mentioned above against p-ERK and ERK as controls. (C) Quantitation of (B) normalized conditions mentioned above against p-ERK and ERK as controls. (C) Quantitation of (B) normalized to to ERK and expressed as a fold difference from the luciferase control. Data are the mean ± SEM of ERK and expressed as a fold difference from the luciferase control. Data are the mean ± SEM of three three independent experiments. * p < 0.05 indicates statistically significant differences. (D) ECV cells independentwere treated experiments. with DMSO * por< 10 0.05 µM indicates U0126 or statistically100 nM Wortmannin significant (2 h) differences. or left untreated. (D) ECV Cells cells were were treatedthen with lysed DMSO and pull-down or 10 µM U0126assays orfor 100CDC42 nM Wortmanninwere performed. (2 h)Total or leftlysates untreated. were also Cells blotted were for then lysedCDC42 and pull-down as the control. assays The for graph CDC42 is a quantitation were performed. of the gel Total normalized lysates were to total also CDC42 blotted and for expressed CDC42 as the control.as a fold The change graph to the is a DMSO quantitation control. of Data the are gel the normalized mean ± SEM to totalof three CDC42 independent and expressed experiments. as a fold* changep < to0.05 the indicates DMSO control.statistically Data significant are the meandifferences.± SEM (E) of ECV three cells independent were treated experiments. with DMSO, 10* p µM< 0.05 indicatesU0126, statistically or 100 nM significantWortmannin differences. (2 h), or left (untreaE) ECVted. cells Cells were were treated then ly withsed and DMSO, pull-down 10 µ Massays U0126, or 100for nM RAC1 Wortmannin were performed. (2 h), or Total leftuntreated. lysates were Cells also were blotted then for lysed RAC1 and as pull-downa control. The assays graph for is RAC1 a werequantitation performed. of Total the gel lysates normalized were also to total blotted RAC1 for an RAC1d expressed as a control.as a fold Thechange graph to the is DMSO a quantitation control. of the gelData normalized are the mean to ± total SEM RAC1 of three and independent expressed experiments. as a fold change * p < 0.05 to indicates the DMSO statistically control. significant Data are the meandifferences.± SEM of ( threeF) Cells independent were treated experiments. with DMSO control, * p < 0.05 Wort indicatesmannin, statisticallyor left untreated. significant Cells were differences. then lysed and blotted against p-ERK and ERK for control. The graph is a quantitation of the gel (F) Cells were treated with DMSO control, Wortmannin, or left untreated. Cells were then lysed and normalized to ERK and expressed as fold change to the control. Data are the mean ± SEM of three blotted against p-ERK and ERK for control. The graph is a quantitation of the gel normalized to independent experiments. * p < 0.05 indicates statistically significant differences. (G) Cells were ERK and expressed as fold change to the control. Data are the mean ± SEM of three independent treated with DMSO or 100 nM Wortmannin (2 h) or were transfected with luciferase, RHOG, CDC42, experiments. * p < 0.05 indicates statistically significant differences. (G) Cells were treated with DMSO or RAC1 siRNA (72 h). Cells were then lysed and blotted against p-AKT and AKT for the control. (H) or 100Representative nM Wortmannin images (2 h)of ora tube were formation transfected assay with (72 luciferase, h after plating) RHOG, of ECV CDC42, cells ortransfected RAC1 siRNA with the (72 h). Cellsvector were thenalone lysed or with and CDC42-DA blotted against and treated p-AKT with and 100 AKT nM Wortmannin for the control. (2 h). (H ) Representative images of a tube formation assay (72 h after plating) of ECV cells transfected with the vector alone or with CDC42-DA and treated with 100 nM Wortmannin (2 h). Cells 2019, 8, 171 11 of 17

3.6. RHOG/CDC42/RAC1 Leads to Tube Formation Downstream from the PI3K Pathway Contrary to ERK inhibition, treating ECV cells with 100 nM of the speciﬁc PI3K inhibitor Wortmannin led to a decrease in the activation of CDC42 and RAC1, as seen by the pull-down assay (Figure6D,E). Cells treated with Wortmannin also showed a decrease in p-ERK (Figure6F). In addition, knocking down RHOG, CDC42, or RAC1 did not affect PI3K signaling in these cells, as seen by the p-AKT Western blot (Figure6G). The role of PI3K in tube formation in these cells was next examined. Wortmannin treatment led to a complete loss of tube formation (Figure6H). However, treating RAC1-DA-transfected cells with the PI3K inhibitor recovered the tube formation (Figure6H). Collectively, this data suggested that PI3K is upstream from the MAPK pathway and RHO GTPases in these cells, leading to tube formation.

3.7. RHOA/ROCK1/ROCK2 Regulate Tube Formation in an ERK-Independent Manner Finally, we aimed to investigate the role of other RHO GTPases such as RHOA, RHOC, and their downstream effectors, ROCK 1 and 2, in tube formation. RHOA, RHOC, ROCK1, and ROCK2 were knocked down in ECV cells (2 oligos were used for each) and the knockdown efﬁciencies were tested for by Western blot and quantitation (Figure7A–C and Figure S2A). As Figure7 shows, the total tube length and numbers and the number of branching points decreased when the cells were treated by RHOA and ROCK1 and 2 (Figure7D–F) but not RHOC (Figure S2B and S2C). Interestingly, Western blot analysis revealed that the level of expression of p-ERK was not affected by RHOA or RHOC siRNA treatment (Figure7G). These data suggest that, as opposed to the RHOG-CDC42-RAC1 axis of GTPases, the RHOA/ROCK1/2 branch regulates tube formation in ECV cells in an ERK-independent manner. Cells 2019, 8, 171 12 of 17 Cells 2019, 8, x 13 of 18

FigureFigure 7. RHO/ROCK 7. RHO/ROCK regulate regulate tube tube formation formation in in an an ERK-independent ERK-independent manner.manner. (A–C) ECV cells cells were were transfectedtransfected with with the the luciferase luciferase control control siRNA siRNA or or with with two two different different RHOA,RHOA, ROCKROCK 1 and 2 2 siRNA siRNA oligos,oligos, respectively. respectively. The cellsThe cells were were lysed lysed and immunoblottedand immunoblotted by Western by Western blot analysis blot analysis with anti-RHOA, with anti- anti-RHOC,RHOA, anti-RHOC, anti-ROCK1, anti-ROCK1, and anti-ROCK2 and anti-ROCK2 antibodies, antibo anddies, with and anti-actin with anti-actin as the control. as the control. The graphs The are quantitationgraphs are quantitation of the gels of normalized the gels normalized to actin to and actin expressed and expressed as fold as decreasesfold decreases compared compared to theto luciferasethe luciferase control. control. Data are Data the are mean the mean± SEM ± SEM of three of three independent independent experiments. experiments. * *p p< < 0.05 indicates statisticallystatistically significant significant differences. differences. (D (–DF–)F Representative) Representative images images of of ECV ECV tubetube formationformation assay (Scale (Scale bar isbar 100 is 100µm) µm) and and quantitation quantitation of totalof total tube tube length, length, total total tube tube number, number,and and thethe numbernumber of branching points,points, respectively, respectively, after after 24, 24, 48, 48, and and 72 72 h ofh of treatment treatment with with RHOA, RHOA, ROCK ROCK 11 andand 2 siRNA vs. vs. control. control. DataData are theare meanthe mean± SEM ± SEM of of three three independent independent experiments. experiments. * *p p< < 0.050.05 indicates statistically significant significant differencesdifferences with with the the luciferase luciferase control. control. (G ()G Cells) Cells were were transfected transfected with with luciferase,luciferase, RHOA,RHOA, or or RHOC RHOC siRNA,siRNA, lysed lysed and and blotted blotted against against p-ERK p-ERK and and ERK ERK for for control. control. The The gels gels were were quantitated,quantitated, and the the level level of ERKof ERK phosphorylation phosphorylation was was expressed expressed as as the the ratio rati p-ERK/ERK.o p-ERK/ERK. Data Data areare thethe meanmean ±± SEMSEM of of three three independentindependent experiments. experiments. * p *< p 0.05 < 0.05 indicates indicates statistically statistically significant significantdifferences. differences. Cells 2019, 8, 171 13 of 17

4. Discussion During angiogenesis, endothelial cells build a network of new vessels to meet the oxygen and nutrient needs of the growing primary tumor, as well as to provide a route for metastasis, rendering the tumor increasingly malignant [5,36]. Understanding angiogenesis and the signaling pathway that regulate the process is instrumental in anti-angiogenic cancer therapy research. RHOG, a member of the RHO family of GTPases, is a main regulator of the actin cytoskeleton, hence it is expected to play a central role in regulating endothelial cell tube formation during angiogenesis [26,33]. The role of the RHO GTPases RAC1, CDC42, RHOA, and RHOC during angiogenesis has been extensively studied [2,8,23,24]. RHOG has been also established as a regulator of angiogenesis and a necessary component of a signaling cascade (along with CDC42 and RAC1), leading to filopodia formation and subsequent tubular remodeling [37]. In this study, we further examine the cross-talk between RHOG and other members of the RHO family of GTPases, particularly CDC42, RAC1, RHOA, and RHOC in the vascular endothelial cells. We also examine the upstream regulators of RHOG in a tube formation assay. To investigate the role of RHOG during angiogenesis, the effect of knocking down RHOG in a tube formation assay was examined in vascular endothelial cells (ECV cells). We find the formation of blood vessels in these cells to be RHOG-dependent. Testing the effect of RAC1 and CDC42 knockdowns reveal that both CDC42 and RAC1 are also required for tube formation in these cells. This is consistent with several reports that previously established the role of CDC42 and RAC1 in angiogenesis [9,23,38]. When dominant active CDC42 or RAC1 constructs are expressed in the RHOG siRNA-transfected cells, the cells regain their ability to form tubes, showing that RHOG regulates tube formation through the CDC42/RAC1 pathway and that RHOG and CDC42/RAC1 are in the same pathway, rather than signal in parallel. This is further directly shown through the direct inhibition of the RHOG siRNA of CDC42 and RAC1 activation in a pull-down assay. This study also shows that CDC42 is upstream from RAC1 in these cells. While knocking down RAC1 did not have an effect on CDC42 activation, the reverse was not true. In addition, in cells co-transfected with RHOG siRNA along with the dominant active CDC42 construct RAC1 activation was recovered (consistent with the tube formation recovery). In fact, in these samples, RAC1 activation appeared to be slightly higher than the control, potentially due to the constitutive exaggerated activity of CDC42-DA (as confirmed by the CDC42 pull-down assay). CDC42 has been indeed previously reported to be upstream from RAC1 in many cell types [39,40]. In addition to the CDC42 dominant active construct, we also used siRNA against the known CDC42/RHOA GAP STARD13 to further manipulate the level of CDC42 activation in these cells [41]. Whereas in other cell types STARD13 is a GAP for CDC42 and RHOA [42–44], in vascular endothelial cells it appears to only inhibit CDC42. An increase in RAC1 activation, compared to control, is also seen in cells transfected with STARD13 siRNA, potentially due to the increase in CDC42 activation described above. Consistently, this is also reflected in the increase in tube formation compared to control cells. Collectively, this data shows that RHOG activates CDC42, which in turn activates RAC1, in this pathway (Figure8-model). Our data are consistent with studies that place RHOG upstream from CDC42/RAC1 in other cells [28,31,33,37,45]. In fact, an earlier study found RHOG to signal upstream from RAC1/CDC42 in endothelial cells leading to filopodia formation essential for tube remodeling [37]. Other studies suggest that RHOG signals in parallel to RAC1 and CDC42 [27]. In fact, RHOG was found in those studies to be activated by RAC1-specific GEFs, such as VAV2, VAV3, and TRIO [46,47], suggesting RHOG acts in parallel to RAC1. This contradiction in reporting could be simply due to different cell types and systems, as has been repeatedly seen for RHO GTPases [48]. Cells 2019, 8, 171 14 of 17 Cells 2019, 8, x 15 of 18

Figure 8. SuggestedSuggested model model based based on on the the data data presented presented in this in this study: study: RHOG RHOG positively positively regulates regulates tube tubeformation formation assay assayin ECV in cells ECV through cells through a CDC42-RAC1-ERK a CDC42-RAC1-ERK.. PI3K appears PI3K appearsto be an upstream to be an upstreamregulator regulatorof RHO GTPases of RHO GTPasesleading to leading tube formation. to tube formation. RHOA and RHOA C seem and to C seemhave todifferent have different roles in rolestube information. tube formation. While RHOA While leads RHOA to positive leads to regulati positiveon regulationof the process of thethrough process ROCK1/2 through as ROCK1/2 downstream as downstreameffectors, RHOC effectors, does not RHOC seem doesto be notnecessary seem toforbe tube necessary formation. for Finally, tube formation. RHOA, unlike Finally, the RHOG- RHOA, unlikeCDC42-RAC1 the RHOG-CDC42-RAC1 pathway, does not pathway, seem to does exert not it seems effect to exerton tube its effect formation on tube through formation the through MAPK thepathway. MAPK pathway.

The role ofof thethe MEK/ERKMEK/ERK pathway pathway in the regulati regulationon of vascular development and angiogenesis isis establishedestablished [[35].35]. Our Our data confirm confirm this role throughthrough thethe inhibitioninhibition ofof tubetube formationformation followingfollowing treatmenttreatment ofof ECVECV cellscells with with the the specific specific MEK MEK inhibitor inhibitor U0126. U0126. While While classically classically MAPKs MAPKs are are thought thought to regulateto regulate RHO RHO GTPases GTPases [49], ERK[49], seemsERK toseems act downstream to act downstream from the RHOG/CDC42/RAC1from the RHOG/CDC42/RAC1 pathway inpathway these cells.in these Indeed, cells. Indeed, knocking knocking down RHOG, down RHOG, CDC42 CDC42 or RAC1 or leadsRAC1 toleads a decrease to a decrease in the in level the oflevel ERK of ERK phosphorylation, phosphorylation, comparable comparable to inhibitionto inhibition with with U0126 U0126 (U0126 (U0126 treatment treatment did did notnot affect CDC42 or RAC1 activation). activation). In In a aprevious previous study study from from our our laboratory, laboratory, we we observed observed an anincrease increase in inERK ERK phosphorylation phosphorylation in inresponse response to to STARD13 STARD13 knoc knockdownkdown in in astrocytoma astrocytoma cells, cells, also also placing ERK downstream fromfrom CDC42CDC42 [[42].42]. In addition, overexpressing dominantdominant activeactive CDC42CDC42 oror RAC1 did not rescue thethe U0126-mediatedU0126-mediated inhibition of tubetube formation,formation, again placing ERK downstreamdownstream from thethe RHOG/CDC42/RAC1 pathway pathway in in these these cells. cells. Another important regulator of angiogenesis and of RHO GTPases is phosphatidylinositol-3- kinase (PI3K) [[16,39,50].16,39,50]. Tube formationformation isis PI3K-dependentPI3K-dependent in these cells.cells. However, contrary to thethe effect on ERK, knocking down RHOG, CDC42, or RAC1 does not affect PI3K signaling in these cells. ThisThis suggestssuggests that, that, contrary contrary to to MAPK, MAPK, RHO RHO GTPases GTPase ares notare upstreamnot upstream of PI3K of inPI3K this pathway.in this pathway. Indeed, cellsIndeed, treated cells with treated Wortmannin with Wortmannin show a decrease show a decrease in p-ERK in and p-ERK in the and activation in the activation of CDC42 of and CDC42 RAC1 and by pull-downRAC1 by pull-down assay, placing assay, PI3K placing upstream PI3K ofupstream the RHOG-CDC42-RAC1-ERK of the RHOG-CDC42-RAC1-ERK axis during axis tube during formation tube information these cells. in these Consistently, cells. Consistently, treating RAC1-DA-transfected treating RAC1-DA-transfected cells with the cells PI3K with inhibitor the PI3K recovered inhibitor the tuberecovered formation. the tube This suggestedformation. that, This similarly suggested to RHOG, that, RAC1similarly activation to RHOG, is downstream RAC1 activation from PI3K is anddownstream directly activating from PI3K tube and formation directly abrogating activating the tube need formation for PI3K signaling.abrogating the need for PI3K signaling.Finally, RHOA, RHOC and their downstream effectors ROCK1 and 2 have been established as regulatorsFinally, of angiogenesisRHOA, RHOC [2, 8and,10, 25their]. Our downstream data confirm effe thectors positive ROCK1 role and of RHOA/ROCK1/ROCK22 have been established inas tuberegulators formation. of angiogenesis Surprisingly, [2,8,10,25]. RHOC shows Our data a negative confirm effect the onpositive tube formation role of RHOA/ROCK1/ROCK2 since the knockdown ofin RHOCtube formation. led to a notable Surprisingly, increase RHOC in tube shows formation. a negative This is ineffect accordance on tube with formation previous since reports the whichknockdown described of RHOC antagonistic led to a notable roles of increase the 2 isoforms in tube formation. RHOA and This RHOC, is in accordance albeit during with cancer previous cell invasionreports which [14]. described The effect antagonistic of RHOA/ROCK1/ROCK2 roles of the 2 isoforms does notRHOA seem and to RHOC, be mediated albeit throughduring cancer ERK. Incell addition, invasion RHOG [14]. The knockdown, effect of RHOA/ROCK1/ROCK2 CDC42 knockdown, and does STARD13 not seem knockdown to be mediated did not have through any effectERK. In addition, RHOG knockdown, CDC42 knockdown, and STARD13 knockdown did not have any effect on RHOA activation. This together suggests that, during tube formation, the RHOG-CDC42- Cells 2019, 8, 171 15 of 17 on RHOA activation. This together suggests that, during tube formation, the RHOG-CDC42-RAC1 pathway and the RHOA-ROCK1/2 pathway operate in parallel and do not compensate for one another. It also suggests that, as opposed to the RHOG-CDC42-RAC1 axis of GTPases, the RHOA/ROCK1/2 branch regulates tube formation in ECV cells in an ERK-independent manner (Figure8). In conclusion, the data in this paper describes, for the first time, the role of RHOG signaling during angiogenesis in vascular endothelial cells. It also dissects its cross-talk with other RHO GTPases and regulation by PI3K. While the mechanism of activation of CDC42/RAC1 by RHOG, the MAPK by RAC1, as well as the intersection with the RHOA/ROCK1/2 pathways remain topics for future work, we believe this study sheds the light on RHOG as a potentially promising target for anti-angiogenesis in cancer therapeutics.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/8/2/171/s1, Figure S1: STARD13/CDC42 and RHOG do not regulate RHOA in ECV cells, Figure S2: Tube formation is not RHOC-dependent. Author Contributions: The following describes the contribution of each author to the work: O.E.A. performed the methodology, software, data analysis and validation as well as original draft preparation. A.F. also performed the methodology, software, data analysis and validation. Both O.E.A. and A.F. contributed equally to the study. M.E.-S. performed the conceptualization of the study, investigation, formal analysis, resources, data curation, as well as writing, review and editing. Funding: This work was funded by intramural funds at the Lebanese American University. Acknowledgments: The authors would like to thank Jonathan Backer and Louis Hodgson (Albert Einstein College of Medicine) for providing constructs. This work was supported by the Natural Science Department at the Lebanese American University and by the School of Arts and Science Research and Development Council (SRDC). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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