PKCα-Mediated Downregulation of RhoA Activity in Depolarized Vascular Smooth Muscle: Synergistic Vasorelaxant Effect of PKCα and ROCK Inhibition

Cell Physiol Biochem 2019;52:76-93 DOI: 10.33594/000000006 Published online: 18 February 2019 76 Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry © 2019 The Author(s). Published by Cell Physiol Biochem Press GmbH&Co. KG González-Montelongo et al.: Functional Role of PKCα-Induced RhoA Inhibition Original Paper Accepted: 14 January 2019 This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission. DOI: 10.33594/000000006 Published online: 18 February 2019 © 2019 The Author(s) Published by Cell Physiol Biochem Press GmbH&Co. KG, Duesseldorf www.cellphysiolbiochem.com PKCα-Mediated Downregulation of RhoA Activity in Depolarized Vascular Smooth Muscle: Synergistic Vasorelaxant Effect of PKCα and ROCK Inhibition María del Carmen González-Montelongoa,b Cristina Porras-Gonzáleza,b Rafaela González-Montelongoa Gonzalo Revilla-Gonzáleza María Dolores Pastora,c Antonio Castellanoa,b Juan Ureñaa,b aInstituto de Biomedicina de Sevilla (IBiS)/Hospital Universitario Virgen del Rocío/CSIC/ Departamento de Fisiología Médica y Biofísica, Universidad de Sevilla, Sevilla, Spain, bRed de Investigación Cardiovascular (RIC)/CIBERCV, Sevilla, Spain, cProteomics Service-Instituto de Biomedicina de Sevilla (IBiS), Sevilla, Spain Key Words Protein kinase C (PKC) • Rho (Rho GTPase) • Vascular smooth muscle cells • Calcium channel • Cell signaling • Phorbol ester • Depolarization Abstract Background/Aims: Protein kinase C (PKC)- and RhoA/Rho-associated kinase (ROCK) play important roles in arterial sustained contraction. Although depolarization-elicited RhoA/ROCK activation is accepted, the role of PKC in depolarized vascular smooth muscle cells (VSMCs) is a subject of controversy. Our aim was to study the role of PKC in arterial contraction and its interaction with RhoA/ROCK. Methods: Mass spectrometry was used to identify the PKC isoenzymes. PKCα levels and RhoA activity were analyzed by western blot and G-LISA, respectively, and isometric force was measured in arterial rings. Results: In depolarized VSMCs RhoA and PKCα were translocated to the plasma membrane, where they colocalize and coimmunoprecipitate. Interestingly, depolarization-induced RhoA activation was downregulated by PKCα, effect reverted by PKCα inhibition. Phorbol 12,13-dibutyrate (PDBu) induced the translocation of PKCα to the plasma membrane, increased the level of RhoA in the cytosol and reduced RhoA/ROCK activity. These effects were reverted when PKC was inhibited. Pharmacological or siRNA inhibition of PKCα synergistically potentiated the vasorelaxant effect of RhoA/ROCK inhibition. Conclusion: The present study provides the first evidence that RhoA activity is downregulated by PKCα in depolarized and PDBu treated freshly isolated VSMCs and arteries, with an important physiological role on arterial contractility. Juan Ureña and Antonio Castellano Instituto de Biomedicina de Sevilla, Edificio IBiS. Campus Hospital Universitario Virgen del Rocío, Avenida Manuel Siurot s/n, E-41013, Sevilla (Spain) Tel. (34)-955-923060, (34)-955-923059, E-Mail [email protected]; [email protected] M. González-Montelongo and C. Porras-González contributed equally to this work. © 2019 The Author(s). Published by Cell Physiol Biochem Press GmbH&Co. KG Cell Physiol Biochem 2019;52:76-93 DOI: 10.33594/000000006 Published online: 18 February 2019 77 Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry © 2019 The Author(s). Published by Cell Physiol Biochem Press GmbH&Co. KG González-Montelongo et al.: Functional Role of PKCα-Induced RhoA Inhibition Introduction Vascular smooth muscle cells (VSMCs) contraction is regulated by the cytosolic Ca2+ concentration and by the Ca2+ sensitivity of myofilaments: the former activates myosin light chain (MLC) kinase and the latter is achieved partly by inhibition of myosin light chain phosphatase (MLCP) and enhanced MLC phosphorylation [1, 2]. Such changes in Ca2+ sensitivity, that participate in the maintenance of arterial contraction, are now known to involve multiple cell signaling processes, including translocation of proteins from the cytosol to the plasma membrane and activation of kinases such as protein kinase C (PKC) and RhoA/ Rho-associated kinase (ROCK). In VSMCs Ca2+ and diacylglycerol (DAG)-dependent (PKCα, PKCβ and PKCγ), Ca2+-independent (PKCε, PKCq, PKCδ and PKCη) and atypical (PKCζ and PKCι/λ) PKC isoenzymes have been described, although their physiological importance in vascular function is still subject of debate [3, 4]. Rho family GTPases are key regulators in a wide range of physiological processes, including VSMCs contraction, cell motility, cell division, etc. [5]. A currently accepted model of Ca2+ sensitization includes inhibition of MLCP by RhoA/ROCK through the MLCP regulatory subunit (MYPT1) and PKC-potentiated inhibitory protein of 17-kDa (CPI-17) [6-8]. Both Ca2+ sensitization pathways not only work in parallel to inhibit MLCP, but they are also known to interact. For instance, RhoA can activate PKCα [9], while, on the other hand, PKC can inhibit or activate RhoA [10, 11]. In addition to GPCR activation, Ca2+ sensitization can be triggered by stimuli that cause membrane depolarization. High extracellular K+ (KCl), a depolarizing solution traditionally used to facilitate Ca2+ influx through L-type Ca2+ channels (LTCCs), can also activate Ca2+ sensitization and sustained contraction via the RhoA/ROCK pathway [12-16]. However, although depolarizationinduced RhoA/ROCK activation is widely accepted, whether Ca2+ sensitization can occur via KCl-induced PKC activation remains unclear. Some reports have suggested that KCl does not cause PKC activation [17], whereas other studies have described that PKC can play a role in Ca2+ sensitization during depolarization [18]. As in some pathological situations VSMCs are depolarized and arterial contractility is increased [19-22], new studies should be addressed to test the role of PKC in depolarized VSMCs, as well as to identify possible new crosstalk between PKC and RhoA/ROCK. In this regard, previous studies in our laboratory have suggested that LTCCs are involved in the maintenance of arterial contraction via the metabotropic release of Ca2+ from internal stores and RhoA/ROCK activation [15, 23]. Since the G protein/phospholipase C cascade can also produce DAG, an endogenous PKC activator, in this work we have examined the role of this protein on depolarization- or phorbol esterinduced sustained arterial contraction and the interaction between PKC and RhoA/ROCK Ca2+ sensitization pathways. We report an unknown PKCα-dependent RhoA/ROCK regulatory mechanism with an important physiological role on arterial contractility. Materials and Methods Animals and Ethical Approval Male wistar rats (250-300 g) were killed with an overdose of sodium thiopental according to the DIRECTIVE 2010/63/EU regarding the use of animals for experimental and other scientific purposes (Royal Decree 53/2013, of February 8th). Experimental procedures were approved by the Department of Animal Production and Experimentation of the Instituto de Biomedicina de Sevilla/Hospital Universitario Virgen del Rocío. Tension Measurements Arterial contractility was performed as previously described [15]. In some experiments endothelial cells were removed by gently rubbing off the intimal surface with cotton-covered wire. The absence of functional endothelium was assessed by determining the lack of effect of acetylcholine (0.5 µM) to induce relaxation in endothelin-I (10 nM) pre-contracted arteries. Cell Physiol Biochem 2019;52:76-93 DOI: 10.33594/000000006 Published online: 18 February 2019 78 Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry © 2019 The Author(s). Published by Cell Physiol Biochem Press GmbH&Co. KG González-Montelongo et al.: Functional Role of PKCα-Induced RhoA Inhibition Preparation of Cytosolic and Membrane or Total Fractions and Western Blot Analysis Given that measurement of proteins in the cytosolic and membrane fractions requires a relatively large amount of tissue, we performed these experiments using aorta and femoral arteries. Immediately after application of the different treatments, arterial rings were immersed in liquid nitrogen and then homogenized in lysis buffer (10 mM Tris-HCl, 250 mM sucrose, 2 mM EDTA, 1 mM DTT, 5 mM MgCl2, 10 µL/mL Na3VO4, 10 µL/mL protease inhibitor cocktail, 10 µL/mL phenylmethylsulfonylfluoride (PMSF), pH 7.4) using a Sample Grinding Kit (GE Healthcare). Samples were centrifuged at 16,000 X g for 15 min at 4°C. This procedure was repeated two or three times until a clarification of the supernatant was obtained. Finally, the supernatant was ultracentrifuged at 100,000 X g for 60 min at 4°C. The supernatant of the ultracentrifugation, containing the cytosolic fraction, was collected and stored frozen until final analysis. To obtain the membrane fraction, the pellet obtained in the ultracentrifugation was washed in membrane lysis buffer (50 mM HEPES, 50 mM NaCl, 1 mM MgCl2, 2 mM EDTA, 1 mM PMSF, 10 mM NaF, 1 mM DTT, 500 µM Na3VO4, and 10 μL/mL protease inhibitors, pH 7.5), and then ultracentrifuged at 120,000 X g for 50 min at 4°C. The final pellet was resuspended in 20 µL membrane lysis buffer in the presence of 1% Triton. To obtain total fractions, a protocol similar to that used for the cytosolic fraction was followed, except for the composition of the lysis buffer (150 mM NaCl, 1mM PMSF, 0.026 mg/mL proteases inhibitors, 20 µL/mL phosphatase inhibitors, 1% Triton and 50 mM Tris-HCl, pH 7.5), and centrifugation at 16,000 X g for 10 min. An aliquot of each protein extract was used for protein quantification by the Bradford Method. After addition of loading buffer (LB: 250 mM Tris, 4% SDS, 25% glycerol, 5% β-mercaptoethanol and 1 mg/mL bromophenol blue), samples were boiled at 95°C for 5 min. Equal amounts of protein were loaded, electrophoresed on 8-12% SDS-PAGE, and transferred to Hybond-P membranes (GE Healthcare). Immunodetection on the western blots was carried out first by membrane preincubation in 5% blotting-grade blocker (nonfat dry milk) in phosphate-buffered saline (PBS: 137 mM NaCl, 2.68 mM KCl, 4.02 mM Na2HPO4, 1.76 mM KH2PO4) with 0.5% Tween 20 (PBS-T), at room temperature or in 5% BSA in Tris-buffered saline (TBS: 100 mM Tris and 1.5 M NaCl) with 0.01% Tween 20 (TBS-T) at 4°C overnight to study phosphorylated residues (p-MYPT1). Membranes were then incubated with specific monoclonal anti-PKCα (diluted 1:1,000; BD Biosciences), monoclonal anti-RhoA (26C4) (diluted 1:250; Santa Cruz Biotechnology), polyclonal anti-MYPT1 (diluted 1:1,000; Merck) or polyclonal anti-phospho-MYPT1 (Thr850) antibody (diluted 1:500; Merck) and monoclonal anti-α tubulin antibody (diluted 1:10,000; Sigma-Aldrich) in 0.5% nonfat dry milk blotting solution overnight at 4°C or with 0.5% BSA in 0.01% TBS-T solution for phosphorylation studies, with gentle agitation. Following incubation, membranes were washed three times (4, 6 and 8 min each) in PBS-T or TBS-T, and incubated for 1 h at room temperature in nonfat dry milk solution with HRP-linked secondary anti-mouse antibody (diluted 1:10,000; GE Healthcare) or anti-rabbit antibody (diluted 1:2,000; Thermo Scientific). After washing six times in PBS-T or TBS-T (4, 6, 8, 10, 12 and 20 min each), immunoreactive bands were visualized using the Enhanced Chemiluminescence Kit (GE Healthcare). The immunoreactive bands were analyzed using an ImageQuant LAS 4000 mini imaging densitometer (GE Healthcare) by quantifying relative band intensities after subtraction of local background in the vicinity of the bands. The density of each band was evaluated with Molecular Analyst software. α-tubulin was used as loading control and values were normalized to the signals obtained with control tissues. The percentage of MYPT1 phosphorylation (expressed as percentage of total MYPT1) was calculated from densitometric values of MYPT1~P as MYPT1 phosphorylation (%) = ((MYPT1~P / Tubulin) / (total MYPT1 / Tubulin)) x 100. RhoA Activation Assay RhoA activity from femoral arterial rings was determined as previously described [15]. Cytosolic Ca2+ Measurements in Isolated VSMCs Cytosolic [Ca2+] measurements from dispersed VSMCs from femoral arteries were performed as previously described [15]. Immunocytochemistry and Confocal Microscopy Immunocytochemical studies were performed as previously described [15]. Cell Physiol Biochem 2019;52:76-93 DOI: 10.33594/000000006 Published online: 18 February 2019 79 Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry © 2019 The Author(s). Published by Cell Physiol Biochem Press GmbH&Co. KG González-Montelongo et al.: Functional Role of PKCα-Induced RhoA Inhibition Immunoprecipitation Experiments Aorta and femoral arteries from four male rats were isolated as previously described, with some modifications. Arteries were incubated overnight at 4 °C and digested in 1 mL Hank’s solution containing 0.1% (wt/vol) collagenase (Sigma Blend Collagenase Type F), 0.002% (wt/vol) elastase (25 U/mg; CalbioChem) and 1 mg/mL BSA at 37°C for 15 min (aorta) and 4 min (femoral). The partially digested tissue was washed twice with 1 mL of enzyme-free buffer solution. Tissue was then transferred into 4 mL of PSS (pH 7.4) containing (in mM): 140 NaCl, 2.7 KCl, 3.5 MgCl2, 10 HEPES and 10 glucose, and gently dispersed to yield isolated VSMCs. Calcium was gradually added to a final concentration of 2.5 mM during 30 min, and 2 mL of dispersed cells were stimulated with 70 mM KCl for 10 min. Both stimulated and unstimulated cells were centrifuged at 16, 200 X g for 1 min at 4°C. Cells were disrupted in 25 µL lysis buffer with 1% Nonidet-P40 (in mM: 150 NaCl, 1 PMSF, 50 Tris-HCl), 0.026 mg/mL proteases inhibitors, 50 µg/mL DNase, pH 7.5) and incubated in ice during 30 min. The supernatant material was collected and the protein content determined by Bradford Method. Each sample (3-5 µg/µL protein) was subjected to preclearing with 40 µL (50% concentration) of protein G-Sepharose beads equilibrated with Nonidet-P40 Cell Lysis Buffer (Invitrogen) rocked for 15 min and then collected by centrifugation at 2, 300 X g for 2 min at 4°C. The supernatant was incubated with 5 µg anti-RhoA antibody (rabbit polyclonal RhoA (119) S/C-179) and rocked overnight at 4°C. Then 40 µL (50% concentration) of protein G-Sepharose beads were added and rocked for 4 h at 4°C. The beads with bound proteins were then collected by centrifugation at 3, 100 X g for 3 min at 4°C. Protein G-Sepharose were washed in wash buffer (in mM: 150 NaCl and 50 Tris-HCl, pH 7.5) three times and boiled in LB. Proteins from the immunoprecipitates were separated onto 8-15% SDS-PAGE gel and transferred to a PVDF membrane. Immunoblotting was performed using mouse monoclonal 1:500 anti-PKCα antibody or 1:250 mouse monoclonal anti-RhoA antibody. Transfection of Arterial Rings siRNA molecules were intracellularly delivered using the Chariot Protein Delivery reagent (Active Motif). Femoral and aorta rings were incubated in DMEM for 1 h at 37°C. For each transfection, 5 µL de Chariot in 50 µL deionized water were mixed with 3.2 µL of siRNA molecules (25 µM) in 50 µL PBS and incubated at room temperature for 30 min to allow formation of the complexes. Arteries were transferred to a sterile 24-well cell culture plate, overlaid with 200 µL of OPTIMEN, covered with 100 µL of Chariot, gently mixed, and incubated for 1 h at 37°C. Tissues were supplemented with 500 µL DMEM for 2 h at 37°C. Every 24 h the medium was changed. At 72 h arterial rings were mounted in the myograph and functional studies were performed. An in silico study of the siRNAs specificity was performed. siRhoA hybridizes only with RhoA (bases 256 (5’) à 276 (3’) for antisense strand (taGCUGUCGGGACUAUCAAAU), and 258 (3’) à 276 (5’) for sense strand (CGACAGCCCUGAUAGUUUAtt). Similarly, of the known PKC isozymes only the PKCα transcript hybridized with the siPKCα (bases 167 (5’) à 187 (3’) for the antisense strand (gtGGACGCUGUACUUACAAGU), and 169 (3 ‘) à 187 (5 ‘) for the sense strand (CCUGCGACAUGAAUGUUCAtt). All analyzed transcripts are from rat, and were extracted from IPA Ingenuity (IPA®, QIAGEN). In silico analysis was performed using SnapGene® Viewer 2.7.2. (GSL Biotech) [24]. Identification of Proteins From Protein Extracts. In-solution Protein Digestion In-solution digestion was performed as reported elsewhere [25]. LC-MS/MS Peptides were analyzed using EASYnano-LC 1000 on a Q Exactive Plus Orbitrap mass spectrometer (Thermo Scientific). Peptides were separated on a pre-column 75 µm x 2 cm, nanoViper, C18, 3 µm, 100 Å (Acclaim PepMap 100) and analytical column 50 µm x 15 cm, nanoViper, C18, 2 µm, 100 Å (Acclaim PepMap RSLC) at a flow rate of 200 nL/min. Water and ACN, both containing 0.1% formic acid, were used as solvents A and B, respectively. The gradient was started and kept at 10% B for 5 min, ramped to 60% B over 60 min or 120 min, depending on the sample complexity, and kept at 90% B for another 5 min. Operating in the datadependent mode to switch from full MS scan (mass width 200–1800 m/z). For data dependent analysis, the top 10 most abundant ions were analyzed for MS/MS analysis while +1 ions were excluded from MS/ MS analysis, with a normalized collision energy of 32%. The raw data were searched with the Sequest HT node of Proteome Discoverer 1.4. The uniprot_ref_rat database (35, 702 protein entries), was utilized for the searches. Variable modifications of methionine oxidation and Carboxymethylation of cysteines were Cell Physiol Biochem 2019;52:76-93 DOI: 10.33594/000000006 Published online: 18 February 2019 80 Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry © 2019 The Author(s). Published by Cell Physiol Biochem Press GmbH&Co. KG González-Montelongo et al.: Functional Role of PKCα-Induced RhoA Inhibition considered. All protein identification results were filtered to include only high confidence peptides with peptide mass deviation <10 ppm, charge state >2 and a minimum of 2 unique peptides per protein. The mass tolerance of fragments ions was < 0.8 Da. Materials and Reagents GF-109203X (GFX), Chelerythrine (Chelery), HA-1077 dihydrochloride (fasudil), phorbol 12, 13-dibutyrate (PDBu), phorbol 12-myristate 13-acetate (PMA), Y27632, 4-Amino-3-(4-chlorophenyl)-1-(tbutyl)-1H-pyrazolo [3, 4-d]pyrimidine (PP2), DAPI dilactate, formalin solution, mouse monoclonal anti-α- tubulin and salts were obtained from Sigma-Aldrich. U-46619 was obtained from Cayman Chemical. RhoA G-LISA was purchased from Cytoskeleton. Sample Grinding Kit, Hybond-P, ECL Western Blotting Analysis System and ECL mouse IgG HRP-linked whole Ab were obtained from GE Healthcare. The rabbit polyclonal anti-RhoA (119) antibody, raised against a peptide mapping within an internal domain of RhoA p21, mouse anti-RhoA (26C4), raised against an epitope corresponding to amino acids 120-150 of RhoA and the phosphatase inhibitors were purchased from Santa Cruz Biotechnology. The mouse anti-PKCα was from BD Biosciences. The anti-rabbit antibody was purchased from Thermo Scientific and anti-mouse was from GE Healthcare. Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 568 goat anti-rabbit IgG, that react with IgG heavy chains and all classes of immunoglobulin light chains from mouse and rabbit respectively, were purchased from Molecular Probes (Invitrogen). Protein levels of phosphorylated-MYPT1 and total MYPT1, were determined using polyclonal anti-p-MYPT1 (Thr850) (Merckmillipore, Cat # 36-003) and anti-MYPT1 (Merckmillipore, Cat #07-672) antibodies, respectively. Fluorescent Mounting Medium was from DAKO. The different siRNA for RhoA (siRhoA), PKCα (siPKC) and scramble (siRNA control) were obtained from Ambion (Life Technologies). Sequencing Grade Modified Trypsin was purchased from Promega. Acclaim PepMap®100, 75 µm x 2 cm, nanViper C18, 3 µm, 100 Å Precolumn and PepMap® RSLC C18, 2 µm, 100 Å, 75 µm x 50 cm Column were purchased from Thermo Scientific. Oasis® HLB 1cc (10 mg) and Oasis® MCX 1 cc (30 mg) Extraction Cartridges were obtained from Oasis (Waters). Ingenuity Pathways Analysis (IPA) The functional and network analyses were generated through the use of QIAGEN´s Ingenuity Pathway Analysis software [26] (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity). The data set from the LC-MS/MS, which used Z-score for assessing the match of observed and predicted up/down regulation patterns. We found that a Z-score is particularly suited for this kind of problem since it serves as both a significance measure and a predictor for the activation state of the regulator. To create protein networks, the proteins were overlaid onto a global molecular network developed from information contained in the Ingenuity Knowledge Base. Data Analysis and Statistics Data are presented as mean ± S.E.M. Differences between sample means were assessed by the one-way ANOVA followed by Tukey post hoc test when comparing three or more groups and two-tailed Student’s t-test to compare two groups. We used non-parametrical statistical tests, Kruskal-Wallis test followed by Mann-Whitney U test, where appropriate. Comparisons between experiments from the same animals were assessed by paired t test. Dose-response curves were fitted to a logistic equation using nonlinear regression analysis tools provided in SigmaPlot software. Values of P < 0.05 were considered significant. Statistical analysis was performed with SPSS software. Results Depolarization-induced RhoA Activation is Regulated in a PKCα-dependent Manner To study the role of PKC in VSMCs contraction, we have treated arterial tissue with two stimuli: 70 mM external K+ (70K) to depolarize membrane potential, and phorbol 12, 13-dibutyrate (PDBu) to activate PKC. Initially, we performed mass spectrometry experiments and IPA analysis (IPA®, QIAGEN) to identify the specific proteins signature for each stimulus in arterial tissue (Fig. 1A). PKCα, PKCβ and PKCδ isoenzymes were upregulated in 70K-induced depolarization, whereas PDBu upregulated PKCα and PKCη Cell Physiol Biochem 2019;52:76-93 DOI: 10.33594/000000006 Published online: 18 February 2019 81 Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry © 2019 The Author(s). Published by Cell Physiol Biochem Press GmbH&Co. KG González-Montelongo et al.: Functional Role of PKCα-Induced RhoA Inhibition Fig. 1. Analysis of the role of PKCα in depolarized arteries. (A) Venn diagram indicating the number of proteins differentially regulated after exposure to control, 70K or PDBu (0.2 µM) for 10 minutes (data from IPA analysis). (B) Venn diagram representing up-regulated (more than two folds) PKC isoenzymes detected in 70K vs. control and PDBu vs. control in rat aorta and femoral arteries. The cutoff for the threshold fold change differences were ≥ 2.0 for upregulation and ≤ 0.5 for downregulation with Z-score between +2 and -2. (C) PKCα distribution (PKCα, green; cell nucleus, blue) in a femoral VSMC in control and after stimulation with 70K for 5 and 10 min. Scale bar, 8 µm. (D) Fluorescence intensity analyses (averaged peripheral (Fm):cytosolic (Fc), Fm/Fc) for different Z sections in control and depolarized conditions (n = 15-20 cells of different independent experiments). Representative immunoblots and analyses of PKCα in the cytosolic (E) and membrane fractions (F) from arteries incubated in 70K (n = 9-16). (G) Western blot confirming that the antibody detects a band of the expected molecular weight (~ 82 kDa). Molecular weight standards (visible, left columns) for 8 % SDS-PAGE and western blotting (chemiluminescence, right columns) of rat arterial homogenates. Statistical significance was calculated by Kruskal-Wallis test followed by Mann-Whitney U test. *P<0.05, **P<0.01, ***P<0.005 vs. control. Cell Physiol Biochem 2019;52:76-93 DOI: 10.33594/000000006 Published online: 18 February 2019 82 Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry © 2019 The Author(s). Published by Cell Physiol Biochem Press GmbH&Co. KG González-Montelongo et al.: Functional Role of PKCα-Induced RhoA Inhibition (Fig. 1B). As both stimuli activated PKCα, an isoenzyme that is expressed in almost all blood vessels [17, 27], we studied its role in depolarization-induced arterial contraction. Since PKC activation involves translocation of the protein to its target site close to the cell membrane, we performed fluorescence immunocytochemistry studies on freshly isolated VSMCs from femoral arteries following stimulation with 70K. Depolarization for 5 and 10 min induced the translocation of PKCα to the periphery of the VSMCs (Fig. 1, C and D). These results were confirmed by western blot analysis, showing that in 70K-treated tissue there was a reduction of PKCα in the cytosolic fraction (Fig. 1E) with a concomitant increase in the membrane fraction (Fig. 1F). Given that these results were repetitive for the rest of the experiments we will only show PKCα in the cytosolic fraction. Fig. 1G shows western blot results confirming Fig. 2. Effect of PKCα inhibition on depolarization-induced RhoA activation. (A) Representative immunoblot (top) and analysis (bottom) illustrating the effects of PKC and ROCK inhibitors on PKCα protein levels in cytosolic extracts obtained from arterial rings bathed with 70K for 10 min (n = 6). 70K-induced PKCα translocation was reverted with GFX (0.5 µM) or fasudil (1 µM). (B) Representative trace of 70K-evoked contraction (top) and statistical analysis of depolarization-induced RhoA activation (bottom). Arrows indicate the times at which arterial rings were frozen for posterior measurement of RhoA activity with the G-LISA test (n = 10). (C) RhoA activity in response to different stimuli. In the 70K+GFX samples, arteries were preincubated with GFX (0.5 µM) for 30 min, and then exposed to 70K for 10 min. U46619 (1 µM) was used as positive control (n = 5). Statistical significance was calculated by one-way ANOVA or Kruskal-Wallis test followed by either Student-Newman-Keuls t-test, Tukey, or MannWhitney U test, where appropriate. *P<0.05, ***P<0.005 vs. control; #P<0.05 vs.70K. Cell Physiol Biochem 2019;52:76-93 DOI: 10.33594/000000006 Published online: 18 February 2019 83 Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry © 2019 The Author(s). Published by Cell Physiol Biochem Press GmbH&Co. KG González-Montelongo et al.: Functional Role of PKCα-Induced RhoA Inhibition Fig. 3. Depolarization-induced RhoA and PKCα translocation to the periphery of rat arteries and coimmunoprecipitation of both proteins. Confocal immunofluorescence images (top panels) and immunofluorescence analyses (bottom panels) illustrating PKCα and RhoA distribution (PKCα, green; RhoA, red; cell nucleus, blue) in resting conditions (control, A) or 70K (B) in VSMCs from femoral arteries. Analyses were performed across four lines in different parts of the cell (a, b, c and d in merged images). Scale bars represent 8 µm (A) or 4 µm (B), (n = 3). Data represent mean ± S.E.M. (C) Effect of depolarization on PKCα- RhoA interaction from dispersed cells of femoral and aortic arterial tissue. Representative experiment that illustrates association of RhoA with PKCα (n = 3). Immunoprecipitation was performed with an anti-RhoA antibody, and western blots using PKCα (top) or RhoA (bottom) antibodies. (D) Western blot confirming that the antibody detects a band of the expected molecular weight (~ 21 kDa). Molecular weight standards (visible, left columns) for 12% SDS-PAGE and western blotting (chemiluminescence, right columns) of rat arterial homogenates (n = 3). Cell Physiol Biochem 2019;52:76-93 DOI: 10.33594/000000006 Published online: 18 February 2019 84 Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry © 2019 The Author(s). Published by Cell Physiol Biochem Press GmbH&Co. KG González-Montelongo et al.: Functional Role of PKCα-Induced RhoA Inhibition that the antibody detects a band of the expected molecular weight. The bisindolylmaleimide GF-109203X (GFX), a PKC inhibitor [28], significantly reversed the reduction of PKCα in the cytosolic fraction (Fig. 2A). We have used 0.5 µM GFX because this value is close to the IC50 for GFX observed in contractions elicited by PDBu (see below) and it does not affect 70K-induced Ca2+ entry measured at 10 min in depolarized VSMCs (R(F340/380) = 0.83, n = 27 in 70K; R(F340/380) = 0.89, n = 37, in 70K+GFX) (data not shown). HA-1077 dihydrochloride (fasudil, 1 µM), a ROCK inhibitor, suppressed 70K-induced PKCα activation (Fig. 2A), suggesting that ROCK is involved in PKCα activation, as also occurs with some agonists [9]. Since ROCK is a downstream RhoA effector, we studied the role of RhoA in this regulatory pathway by measuring RhoA activity. As expected from our previous results [15, 16], exposure of arterial rings to 70K induced an increase in RhoA activity (Fig. 2B). Interestingly, 0.5 µM GFX, which did not significantly affected basal RhoA activity, markedly potentiated the effect of depolarization (Fig. 2C). An increase in the activity of RhoA was observed when arteries were treated with U46619, a thromboxane A2 analog that activates RhoA (Fig. 2C). As these results suggest that PKCα can negatively regulate RhoA activity, we hypothesized that depolarization could translocate both proteins toward the plasma membrane and facilitate their interaction. We next examined in immunostained isolated VSMCs the cellular distribution of RhoA and PKCα under basal conditions and in response to depolarization. Fig. 3A (top panels) shows the distribution of RhoA and PKCα in an isolated VSMCs under resting conditions. The line graph analysis across four lines in different parts of the cell (a, b, c and d) shows that fluorescence intensity was almost uniform across the cytoplasm (Fig. 3A, bottom panels). Application of 70K for 10 min caused VSMCs contraction and RhoA and PKCα translocation toward the periphery of the cell, reflecting the activation of both proteins (Fig. 3B, top panels). Accordingly, the line graph analysis reflected higher fluorescence intensity in the periphery than in the central cytosol (Fig. 3B, bottom panels), confirming the colocalization of PKCα and RhoA in the cell periphery in the 70K-treated VSMCs. To determine whether depolarization-induced regulation of RhoA by PKCα occurs as a result of their physical interaction, isolated VSMCs were stimulated with 70K for 10 min, and cell extracts were immunoprecipitated with a RhoA antibody. Western blot analyses using monoclonal anti- PKCα or anti-RhoA antibodies show that stimulation with 70K produces an increase in the association of RhoA with PKCα (Fig. 3C). Fig. 3D shows western blot results confirming that the antibody detects a band of the expected molecular weight. Taken together, these data demonstrate that depolarization activates RhoA and PKCα and recruits both proteins to the plasma membrane, where they interact, favoring RhoA inhibition by PKCα. Pharmacologic Inhibition or siRNA-mediated Silencing of RhoA/ROCK and PKCα Produces Synergistic Vasorelaxant Effects on Depolarization-induced Sustained Arterial Contraction To study the physiological role of RhoA/ROCK regulation by PKCα we measured isometric force in arterial rings treated with PKCα and RhoA/ROCK inhibitors or siRNAs. Fig. 4A shows a representative trace illustrating the cumulative vasorelaxant effect of fasudil on femoral arterial rings precontracted with 70K. Unexpectedly, GFX, that had no effect on depolarization-induced contraction (Fig. 4B), markedly potentiated, in a synergistic and nonadditive manner, the vasorelaxant effect of fasudil (IC50 = 7.36 µM for fasudil, IC50 = 3.30 µM for GFX+fasudil) (Fig. 4, C and D). As RhoA/ROCK also regulates signaling in endothelial cells [29], we performed experiments in endothelium-denuded femoral arterial rings. In these arteries, GFX (0.5 µM) markedly potentiated the vasorelaxant effect of fasudil on sustained contraction, indicating that the endothelium is not involved in this phenomenon (IC50 = 4.8 µM for fasudil, n= 9; IC50 = 3.4 µM for GFX+fasudil, n = 6) (data not shown). To eliminate nonspecific pharmacological effects of ROCK and PKC inhibitors, we transiently knocked-down RhoA and PKCα with specific and validated siRNAs (see Material and Methods). Consistent with the results obtained in the pharmacological approach, application of siRhoA significantly reduced (~17%) the sustained component of 70K-induced contraction (Fig. 4, E and H), when compared to a control siRNA (scramble), whereas siPKCα did not produce any significant Cell Physiol Biochem 2019;52:76-93 DOI: 10.33594/000000006 Published online: 18 February 2019 91 Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry © 2019 The Author(s). Published by Cell Physiol Biochem Press GmbH&Co. KG González-Montelongo et al.: Functional Role of PKCα-Induced RhoA Inhibition by GFX. However, in depolarized arteries we cannot rule out the participation of PKCβ and PKCd, as these isoenzymes are also activated in addition to PKCa. Although it is known that cAMP and cyclic GMP-dependent kinases (PKA and PKG, respectively) inhibit RhoA-dependent functions in agonist treated VSMCs [34], the new crosstalk between PKCα and RhoA/ROCK in depolarized VSMCs described in this report represents a newly identified mechanism involved in the regulation of Ca2+ sensitization in VSMCs. Our results do not distinguish between the possibility that association of PKCα with RhoA can occur directly or whether it is mediated through a molecular link. Evidences for a direct protein-protein interaction between certain PKC isoenzymes and Rho GTPases that results in RhoA activation have been described previously [35]. On the other hand, RhoA could also associate with other molecules forming a complex. Consistent with this idea, it has been described that HSP27 can be a potential molecular link between RhoA and PKCα [36]. Further experiments are necessary to analyze the molecular basis of the interaction between PKCα and RhoA in VSMCs. Physiological Significance of the Present Findings Our data support a working model including a new crosstalk between PKCα and RhoA/ ROCK with an important physiological role on sustained arterial contraction (Fig. 8). Fig. 8A shows that PDBu-induced contraction could be mediated by PKCα activation and MLCP inhibition (pathway 1). As PKCα also reduced RhoA/ROCK activity (pathway 2), arterial contraction induced by RhoA/ROCK (pathway 3) is not important in this process. It could explain, the lack of effect of ROCK inhibitors on contraction in phorbol-ester treated arteries described here (Fig. 6D and E) and reported by other laboratories [37]. In depolarized arteries, both RhoA/ROCK and PKCα are activated, leading to MLCP inhibition and contraction (pathways 3 and 1, respectively). Our data show that RhoA/ROCK is the most important pathway under this stimulus and that their function is negatively regulated by PKCα (pathway 2). In vitro experiments indicate that GFX increases RhoA activity (Fig. 2C), which should increase contraction through pathway 3. However, functional experiments show that GFX has no effect on depolarization-induced contraction (Fig. 4B). This could reflect that potentiation of contraction through pathway 3 is counteracted by the vasorelaxation induced by the inhibition of pathway 1 and the negative feedback of ROCK on RhoA through pathway 4 and 2. The synergistic vasorelaxant effect of GFX and fasudil in depolarized and PDBu-treated arteries could be a consequence of the fact that the presence of PKCα inhibitors facilitates RhoA/ROCK function and increases the vasorelaxant effect of fasudil. In contrast, in depolarized arteries, the presence of PDBu, that activates PKCα and reduces RhoA activity, suppressed the vasorelaxant effect of fasudil (Fig. 5B). In conclusion, although further studies are needed to determine the role of Ca2+ sensitization in vascular pathophysiology, the combined use of ROCK and PKCα inhibitors might be an attractive therapeutic strategy in the treatment of the arterial vasospasm present in some cardiovascular diseases [38, 39]. Acknowledgements This work was supported by the “Red de Investigación Cardiovascular, RIC, RD12/0042/0041” of the Instituto de Salud Carlos III and by Ministerio de Economía y Competitividad and FEDER (SAF2013-46806-R and SAF2017-89474-R). We thank Dr. Konstantin Levitsky (Instituto de Biomedicina de Sevilla) for technical help with the confocal microscopy. The authors acknowledge support from Supercomputing and Bioinformatics Center at Malaga University to carry out the studies in IPA. Cell Physiol Biochem 2019;52:76-93 DOI: 10.33594/000000006 Published online: 18 February 2019 92 Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry © 2019 The Author(s). 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