Abstract 59: Loss of Cullin-3 in Smooth Muscle Increases RhoA, Vascular Contraction and Arterial Pressure

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Christopher Pelham ◽  
Pimonrat Ketsawatsomkron ◽  
Justin Grobe ◽  
Frank Faraci ◽  
Curt Sigmund
Keyword(s):  
Smooth Muscle ◽  
Arterial Pressure ◽  
Vascular Function ◽  
Enzyme Inhibitor ◽  
Cyclin E ◽  
Rho Kinase ◽  
Dominant Negative ◽  
Dark Phase ◽  
Kinase Signaling ◽  
Cullin 3

Mutations in Cullin-3 result in early-onset hypertension in humans through an undefined mechanism. Cullin-3 RING E3 ubiquitin ligase complex mediates proteasomal degradation of RhoA, a key mediator of vascular smooth muscle tone. We reported that smooth muscle-specific expression of dominant negative PPARγ (S-P467L) in transgenic mice causes hypertension and aortic dysfunction via increased RhoA/Rho-kinase signaling. Dominant negative PPARγ caused decreased Cullin-3 protein (0.31±0.04-fold of Non-Transgenic (NT); P<0.05) and decreased ratio of active Nedd8-Cullin-3 / Cullin-3 (0.06±0.04 vs 0.51±0.18 NT; P<0.05), which correlated with increased protein levels of Cullin-3 substrates RhoA (2.6±0.2-fold of NT; P<0.05) and Cyclin E (2.0±0.1-fold of NT; P<0.05) in S-P467L medial aorta. We tested the hypothesis that Cullin-3 controls arterial pressure by regulating vascular function. siRNA-mediated knockdown of Cullin-3 (72 hr), confirmed by Western blot, increased RhoA (2.5±0.1-fold of NC1 negative control siRNA; P<0.05) and Cyclin E (1.9±0.2-fold of NC1; P<0.05) protein in primary rat aortic smooth muscle cells. Inhibition of cullin-RING ligase activity using the Nedd8-activating enzyme inhibitor, MLN4924 (1μM, 16 hr), also increased RhoA (4.3±1.0-fold of DMSO vehicle). Treatment of aortic rings from control mice with MLN4924 (1μM in DMEM/F12, 16 hr) resulted in enhanced agonist-mediated contraction in response to endothelin 1 (30nM: 0.35±0.05g vs 0.19±0.02g DMSO vehicle; P<0.05), serotonin (3μM: 1.2±0.07g vs 0.9±0.08g; P<0.05), and phenylephrine (3μM: 1.1±0.05g vs 0.7±0.05g, P<0.05) that was Rho-kinase-dependent, despite decreased contraction to KCl (100mM: 0.7±0.03g vs 1.0±0.03g; P<0.05). Finally, administration of MLN4924 (30mg/kg TID, 2 days) to control mice in vivo increased mean arterial pressure during the light phase (121±4 vs. 108±3mmHg baseline, P<0.05; dark phase: 122±4 vs. 123±4mmHg baseline, NS) as measured by radiotelemetry. Our results demonstrate that interference with PPARγ in smooth muscle causes vascular dysfunction via impaired Cullin-3-mediated regulation of RhoA/Rho-kinase signaling and provide a mechanistic link between mutations in Cullin-3 and hypertension.

Smooth muscle F-actin disassembly and RhoA/Rho-kinase signaling during endotoxin-induced alterations in pulmonary arterial compliance

2004 ◽  
Vol 287 (4) ◽  
pp. L649-L655 ◽  
Author(s):  
Christa Boer ◽  
Geerten P. van Nieuw Amerongen ◽  
A. B. Johan Groeneveld ◽  
Gert Jan Scheffer ◽  
Jaap J. de Lange ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Actin Cytoskeleton ◽  
Vascular Function ◽  
Kinase Inhibitor ◽  
Arterial Compliance ◽  
Pulmonary Arteries ◽  
Rho Kinase ◽  
Kinase Signaling ◽  
Arterial Mechanics ◽  
Pulmonary Arterial

Endotoxemia is associated with changed pulmonary vascular function with respect to vasoreactivity, endothelial permeability, and activation of inducible nitric oxide synthase II (NOSII). However, whether altered passive arterial wall mechanics contribute to this endotoxin-induced pulmonary vascular dysfunction is still unknown. Therefore, we investigated whether endotoxin affects the passive arterial mechanics and compliance of isolated rat pulmonary arteries. Pulmonary arteries of pentobarbital-anesthetized Wistar rats ( n = 55) were isolated and exposed to Escherichia coli endotoxin (50 μg/ml) for 20 h. Endotoxin increased pulmonary artery diameter and compliance (transmural pressure = 13 mmHg) in an endothelium-, Ca2+-, or NOSII-induced NO release-independent manner. Interestingly, the endotoxin-induced alterations in the passive arterial mechanics were accompanied by disassembly of the smooth muscle cell (SMC) F-actin cytoskeleton. Disassembly of F-actin by incubation of control arteries with the cytoskeleton-disrupting agent cytochalasin B or the Rho-kinase inhibitor Y-27632 induced a similar increase in passive arterial diameter and compliance. In contrast, RhoA activation by lysophosphatidic acid prevented the endotoxin-induced alterations in the pulmonary SMC F-actin cytoskeleton and passive mechanics. In conclusion, these findings indicate that disassembly of the SMC F-actin cytoskeleton and RhoA/Rho-kinase signaling act as mediators of endotoxin-induced changes in the pulmonary arterial mechanics. They imply the involvement of F-actin rearrangement and RhoA/Rho-kinase signaling in endotoxemia-induced vascular lung injury.

Abstract 048: Expression of a Hypertension-causing Mutation in Cullin 3 (CUL3Δ9) Specifically in Smooth Muscle Causes Vascular Dysfunction and Hypertension

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Larry N Agbor ◽  
Jing Wu ◽  
Stella-Rita C Ibeawuchi ◽  
Chunyan Hu ◽  
Deborah R Davis ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Transgenic Mice ◽  
Basilar Artery ◽  
Kinase Inhibitor ◽  
Vascular Dysfunction ◽  
Rho Kinase ◽  
Dominant Negative ◽  
Nitric Oxide Donor ◽  
Ang Ii ◽  
Cullin 3

Pseudohypoaldosteronism type II (PHAII) patients with mutations in cullin 3 (CUL3) resulting in exon 9 deletion (CUL3Δ9), exhibit severe early onset hypertension correlated with impaired kidney function. However, the extra-renal mechanisms remain uninvestigated. We hypothesized that expression of CUL3Δ9 protein in smooth muscle in mice impairs endogenous wildtype CUL3 (CUL3-WT) function and causes vascular dysfunction and hypertension. We generated transgenic mice inducibly expressing CUL3Δ9 protein in smooth muscle (S-CUL3Δ9) and measured blood pressure (BP) by radiotelemetry. We assessed vascular responses in the cerebral basilar artery and aorta using a pressurized and a wire myograph, respectively. S-CUL3Δ9 mice exhibited reduced expression of endogenous CUL3WT protein compared to non-transgenic (NT) in aorta. Systolic BP was significantly increased in S-CUL3Δ9 mice (127±2 S-CUL3Δ9 vs 117±1 NT, p=0.02). Basilar artery from S-CUL3Δ9 mice exhibited significantly impaired vasorelaxation to acetylcholine (ACh) (at 100 μM: 15±4% S-CUL3Δ9 vs 65±5% NT, p<0.0001), and to the nitric oxide donor sodium nitroprusside (SNP) (at 100 μM: 59±2% S-CUL3Δ9 vs 90±5% NT, p<0.05). Vasocontraction to angiotensin II (Ang II), phenylephrine (PE) and to endothelin 1 (ET-1) were significantly elevated in S-CUL3Δ9 transgenic mice. Consistent with data from basilar artery, aorta from S-CUL3Δ9 transgenic mice exhibited impaired ACh-mediated relaxation (at 100 μM: 55±2% S-CUL3Δ9 vs 71±7% NT, p<0.0001). Total RhoA protein was significantly elevated in aorta of S-CUL3Δ9 transgenic mice (1.6±0.2 S-CUL3Δ9 vs 1.0±0.1 NT, P<0.05). Serotonin stimulation caused a significant increase in active RhoA in S-CUL3Δ9 aorta (1.83±0.04 S-CUL3Δ9 versus 1.52±0.06 NT, p=0.005). Preincubation with the Rho-kinase inhibitor (Y27632) restored endothelium-dependent relaxation in basilar artery and aorta of S-CUL3Δ9 mice. Ang II infusion via osmotic minipump (200 ng/kg/min) resulted in elevated BP response (Systolic BP: 147 ± 2 S-CUL3Δ9 versus 130 ± 5 NT, p=0.04) and increased aortic stiffening in S-CUL3Δ9 mice. We conclude that CUL3Δ9 acts in a dominant negative manner by interfering with CUL3-WT and contributes at least in part to hypertension via its effects on the vasculature.

Abstract 310: Deoxycorticosterone Acetate (doca)-salt Exacerbates Hypertension and Vascular Dysfunction in Mice Expressing Dominant Negative Peroxisome Proliferator-activated Receptor-gamma (pparg) in Smooth Muscle

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Pimonrat Ketsawatsomkron ◽  
Deborah R Davis ◽  
Aline M Hilzendeger ◽  
Justin L Grobe ◽  
Curt D Sigmund
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Transgenic Mice ◽  
Vascular Function ◽  
Vascular Dysfunction ◽  
Critical Role ◽  
Surrogate Marker ◽  
Dominant Negative ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor

PPARG, a ligand-activated transcription factor plays a critical role in the regulation of blood pressure and vascular function. We hypothesized that smooth muscle cell (SMC) PPARG protects against hypertension (HT) and resistance vessel dysfunction. Transgenic mice expressing dominant negative PPARG (S-P467L) in SMC or non-transgenic controls (NT) were implanted with DOCA pellet and allowed ad libitum access to 0.15 M NaCl for 21 days in addition to regular chow and water. Blood pressure was monitored by telemetry and mesenteric arterial (MA) function was assessed by pressurized myograph. At baseline, 24-hour mean arterial pressure (MAP) was similar between NT and S-P467L mice, while the transgenic mice were tachycardic. DOCA-salt increased MAP to a much greater degree in S-P467L mice (Δ MAP; S-P467L: +34.2±6.0, NT: +13.3±5.7, p<0.05 vs NT). Heart rate was similarly decreased in both groups after DOCA-salt. Vasoconstriction to KCl, phenylephrine and endothelin-1 did not differ in MA from DOCA-salt treated NT and S-P467L, while the response to vasopressin was significantly reduced in S-P467L after DOCA-salt (% constriction at 10-8 M, S-P467L: 31.6±5.6, NT: 46.7±3.8, p<0.05 vs NT). Urinary copeptin, a surrogate marker for arginine vasopressin was similar in both groups regardless of treatment. Vasorelaxation to acetylcholine was slightly impaired in S-P467L MA compared to NT at baseline whereas this effect was further exaggerated after DOCA-salt (% relaxation at 10-5 M, S-P467L: 56.1±8.3, NT: 79.4±5.6, p<0.05 vs NT). Vascular morphology at luminal pressure of 75 mmHg showed a significant increase in wall thickness (S-P467L: 18.7±0.8, NT: 16.0±0.4, p<0.05 vs NT) and % media/lumen (S-P467L: 8.4±0.3, NT: 7.1±0.2, p<0.05 vs NT) in S-P467L MA after DOCA-salt. Expression of tissue inhibitor of metalloproteinases (TIMP)-4 and regulator of G-protein signaling (RGS)-5 transcript were 2- and 3.5-fold increased, respectively, in MA of NT with DOCA-salt compared to NT baseline. However, this induction was markedly blunted in S-P467L MA. We conclude that interference with PPARG function in SMC leads to altered gene expression crucial for normal vascular homeostasis, thereby sensitizing the mice to the effects of DOCA-salt induced HT and vascular dysfunction.

scholarly journals Cullin-3 Regulates Vascular Smooth Muscle Function and Arterial Blood Pressure via PPARγ and RhoA/Rho-Kinase

2012 ◽  
Vol 16 (4) ◽  
pp. 462-472 ◽  
Author(s):  
Christopher J. Pelham ◽  
Pimonrat Ketsawatsomkron ◽  
Séverine Groh ◽  
Justin L. Grobe ◽  
Willem J. de Lange ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Arterial Blood Pressure ◽  
Muscle Function ◽  
Rho Kinase ◽  
Arterial Blood ◽  
Smooth Muscle Function ◽  
Cullin 3

Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors

2005 ◽  
Vol 288 (1) ◽  
pp. G23-G31 ◽  
Author(s):  
Jiean Huang ◽  
Huiping Zhou ◽  
Sunila Mahavadi ◽  
Wimolpak Sriwai ◽  
Vijay Lyall ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Dominant Negative ◽  
Phosphatase Inhibitors ◽  
Sustained Contraction ◽  
Pi Hydrolysis

The signaling cascades initiated by motilin receptors in gastric and intestinal smooth muscle cells were characterized. Motilin bound with high affinity (IC50 0.7 ± 0.2 nM) to receptors on smooth muscle cells; the receptors were rapidly internalized via G protein-coupled receptor kinase 2 (GRK2). Motilin selectively activated Gq and G13, stimulated Gαq-dependent phosphoinositide (PI) hydrolysis and 1,4,5-trisphosphate (IP3)-dependent Ca2+ release, and increased cytosolic free Ca2+. PI hydrolysis was blocked by expression of Gαq minigene and augmented by overexpression of dominant negative RGS4(N88S) or GRK2(K220R). Motilin induced a biphasic, concentration-dependent contraction (EC50 = 1.0 ± 0.2 nM), consisting of an initial peak followed by a sustained contraction. The initial Ca2+-dependent contraction and myosin light-chain (MLC)20 phosphorylation were inhibited by the PLC inhibitor U-73122 and the MLC kinase inhibitor ML-9 but were not affected by the Rho kinase inhibitor Y27632 or the PKC inhibitor bisindolylmaleimide. Sustained contraction and MLC20 phosphorylation were RhoA dependent and mediated by two downstream messengers: PKC and Rho kinase. The latter was partly inhibited by expression of Gαq or Gα13 minigene and abolished by coexpression of both minigenes. Sustained contraction and MLC20 phosphorylation were partly inhibited by Y27632 and bisindolylmaleimide and abolished by a combination of both inhibitors. The inhibition reflected phosphorylation of two MLC phosphatase inhibitors: CPI-17 via PKC and MYPT1 via Rho kinase. We conclude that motilin initiates a Gαq-mediated cascade involving Ca2+/calmodulin activation of MLC kinase and transient MLC20 phosphorylation and contraction as well as a sustained Gαq- and Gα13-mediated, RhoA-dependent cascade involving phosphorylation of CPI-17 by PKC and MYPT1 by Rho kinase, leading to inhibition of MLC phosphatase and sustained MLC20 phosphorylation and contraction.

scholarly journals Gq-dependent signalling by the lysophosphatidic acid receptor LPA3 in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA

2008 ◽  
Vol 411 (3) ◽  
pp. 543-551 ◽  
Author(s):  
Wimolpak Sriwai ◽  
Huiping Zhou ◽  
Karnam S. Murthy
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Lysophosphatidic Acid ◽  
Light Chain ◽  
Rho Kinase ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Sustained Contraction ◽  
C3 Exoenzyme ◽  
In Cells

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA3 receptors, but not LPA1 and LPA2, receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the Gαq, but not the Gαi, minigene. Initial contraction and MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-β (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC20 and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IκBα were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr696 was masked by phosphorylation of MYPT1 at Ser695 by PKA derived from IκB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-β and MLCK and phosphorylation of MLC20, and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC20. Although Rho kinase was activated, phosphorylation of MYPT1 at Thr696 by Rho kinase was masked by phosphorylation of MYPT1 at Ser695 via cAMP-independent PKA derived from the NF-κB pathway.

Abstract 18390: Ang II activates the RhoA Exchange Factor Arhgef1 in Humans

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Maria-Luigia Carbone ◽  
Jérémy Brégeon ◽  
Nabila Devos ◽  
Anne Blanchard ◽  
Michel Azizi ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Rho Kinase ◽  
Renin Angiotensin System ◽  
Muscle Cells ◽  
Ang Ii ◽  
Kinase Signaling ◽  
Angiotensin System ◽  
Normotensive Subjects

Introduction: Although a causative role for RhoA-Rho kinase signaling has been recognized in the development of human hypertension, the molecular mechanism(s) as well as the RhoA exchange factors (GEFs) responsible for the over activation of RhoA remain unknown. Arhgef1 has been recently identified as a RhoA GEF involved in Ang II-mediated regulation of vascular tone and hypertension in mice. Hypothesis: Here we assessed the hypothesis that Arhgef1 is activated and involved in the activation of RhoA-Rho kinase signaling by Ang II in humans. Methods: We used in vitro stimulation of human coronary artery smooth muscle cells and human peripheral blood cells (PBMC) by Ang II (0.1 μmol/L), and PBMC isolated from normotensive subjects before and after activation of the renin-angiotensin system by a low-salt diet for 7 days (checked by the increase in plasma aldosterone and active renine). Activation of Arhgef1 was monitored by measuring its tyrosine phosphorylation by western blot and phosphorylation of the Rho kinase target MYPT was used to measure the activity of RhoA/Rho kinase signaling. Results: In vitro, Ang II induced activation of Arhgef1 in human vascular smooth muscle cells and PBMC. Silencing of Arhgef1 expression by siRNA in human vascular smooth muscle cells inhibited Ang II-induced activation of RhoA-Rho kinase signaling (0.67±0.12 vs 3.21±0.91relative to unstimulated condition, n=4, P<0.05). In normotensive subjects, activation of the renin-angiotensin system by a low-salt diet stimulated Arhgef1 activity in PBMC (1.26±0.12-fold over basal level, n=39, P<0.05). This activation was associated with an increase activity of RhoA-Rho kinase signaling (1.33±0.14-fold over basal level, n=39, P<0.05). Conclusions: Our results show that Ang II stimulates Arhgef1 activity and strongly suggest that Arhgef1 mediates Ang II-induced RhoA activation in humans. Moreover, we show for the first time that measurement of RhoA GEF activity in PBMC might be a useful method to evaluate RhoA GEF activity in humans.

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