Cullin-3 Mutations Research Articles

Neuronal knockdown of Cullin3 as a Drosophila model of autism spectrum disorder

Mutations in Cullin-3 (Cul3), a conserved gene encoding a ubiquitin ligase, are strongly associated with autism spectrum disorder (ASD). Here, we characterize ASD-related pathologies caused by neuron-specific Cul3 knockdown in Drosophila. We confirmed that neuronal Cul3 knockdown causes short sleep, paralleling sleep disturbances in ASD. Because sleep defects and ASD are linked to metabolic dysregulation, we tested the starvation response of neuronal Cul3 knockdown flies; they starved faster and had lower triacylglyceride levels than controls, suggesting defects in metabolic homeostasis. ASD is also characterized by increased biomarkers of oxidative stress; we found that neuronal Cul3 knockdown increased sensitivity to hyperoxia, an exogenous oxidative stress. Additional hallmarks of ASD are deficits in social interactions and learning. Using a courtship suppression assay that measures social interactions and memory of prior courtship, we found that neuronal Cul3 knockdown reduced courtship and learning compared to controls. Finally, we found that neuronal Cul3 depletion alters the anatomy of the mushroom body, a brain region required for memory and sleep. Taken together, the ASD-related phenotypes of neuronal Cul3 knockdown flies establish these flies as a genetic model to study molecular and cellular mechanisms underlying ASD pathology, including metabolic and oxidative stress dysregulation and neurodevelopment.

Abstract 084: Smooth Muscle Cullin-3 Regulates The Renal Baroreceptor Mechanism Via Lamin AC

Cullin-3 (CUL3) is a critical component of the CUL3-Ring-Ligase (CRL) ubiquitin ligase complex. Patients carrying mutations in CUL3 exhibit early onset of hypertension (HTN). We demonstrated that conditional ablation of CUL3 in vascular smooth muscle cells (VSMCs) results in vascular dysfunction, arterial stiffness, and severe HTN in mice. We hypothesized that 1) conditional deletion of CUL3 in VSMCs (S-CUL3KO) results in severe HTN via the renin-angiotensin system (RAS)-dependent mechanism, and 2) S-CUL3KO mice exhibit an impaired baroreceptor mechanism regulating renin. Mice carrying a conditional allele of CUL3 were bred with mice expressing a tamoxifen-inducible CRE-recombinase driven by a smooth muscle promoter (ISM-CRE, control). S-CUL3KO mice exhibited elevated systolic blood pressure (SBP) 3 weeks after tamoxifen administration (S-CUL3KO: 164±1 vs. ISM-CRE: 124±4 mmHg, p<0.05). To test the first hypothesis captopril or candesartan (10 mg/kg/day and 100 mg/kg/day in drinking water, respectively) were administered to S-CUL3KO once severe HTN was already established. Captopril or candesartan treatment for 1 week reversed elevated SBP to 117±4 mmHg (p<0.05) and 127±2 mmHg (p<0.05), respectively, in S-CUL3KO. Despite the severity of HTN in S-CUL3KO, the level of renin mRNA in the kidney was surprisingly unaltered (p>0.05) when compared to the normotensive control mice. The protein expression of lamin AC, a crucial component of the nuclear mechanotransducer controlling renin expression, was significantly increased in S-CUL3KO kidneys (0.11±0.02 vs. 0.05±0.01 normalized RFU, p<0.05). This suggests lamin AC may be a target protein of CUL3-ubiquitin ligase complex. Supporting this concept, treatment of A10 smooth muscle cells with MLN4914, an inhibitor of the Cullin pathway, results in a 2-fold increase in lamin AC expression (0.007±0.001 vs. 0.0154±0.0004, normalized RFU, p<0.05). Furthermore, co-immunoprecipitation of CUL3 in HEK293 cells immunoprecipitated lamin AC. Our findings support the hypothesis that 1) CUL3 regulates RAS via the renal baroreceptor mechanism and 2) lamin AC is a potential target of the CRL3-mediated regulation. Understanding CUL3 target proteins might help the development of new strategies to mitigate HTN.

Cullin 3 Exon 9 Deletion in Familial Hyperkalemic Hypertension Impairs Cullin3-Ring-E3 Ligase (CRL3) Dynamic Regulation and Cycling.

Cullin 3 (CUL3) is the scaffold of Cullin3 Ring E3-ligases (CRL3s), which use various BTB-adaptor proteins to ubiquitinate numerous substrates targeting their proteasomal degradation. CUL3 mutations, responsible for a severe form of familial hyperkalemia and hypertension (FHHt), all result in a deletion of exon 9 (amino-acids 403-459) (CUL3-∆9). Surprisingly, while CUL3-∆9 is hyperneddylated, a post-translational modification that typically activates CRL complexes, it is unable to ubiquitinate its substrates. In order to understand the mechanisms behind this loss-of function, we performed comparative label-free quantitative analyses of CUL3 and CUL3-∆9 interactome by mass spectrometry. It was observed that CUL3-∆9 interactions with COP9 and CAND1, both involved in CRL3 complexes’ dynamic assembly, were disrupted. These defects result in a reduction in the dynamic cycling of the CRL3 complexes, making the CRL3-∆9 complex an inactive BTB-adaptor trap, as demonstrated by SILAC experiments. Collectively, the data indicated that the hyperneddylated CUL3-∆9 protein is inactive as a consequence of several structural changes disrupting its dynamic interactions with key regulatory partners.

Abstract P086: Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, And Hypertension

Mutations in CULLIN3 gene (causing in-frame deletion of exon 9, termed Cul3Δ9) cause human hypertension (HT), which is driven by a combination of renal tubular and vascular mechanisms. We have previously shown that disruption of Cullin3 (CUL3) in vascular smooth muscle impairs nitric oxide (NO) signaling and vasodilation through decreased cGMP bioavailability, strongly supporting a role of vascular CUL3 in blood pressure (BP) regulation. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE ERT2 mice. Four weeks after tamoxifen, the resultant mice (E-Cul3Δ9) developed nocturnal HT (Night time peak systolic BP, E-Cul3Δ9: 135±3 vs Control: 124±3 mmHg) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s). No difference was seen in daytime BP. To determine whether vascular remodeling impairs baroreflex function, we performed power spectral analysis. Heart rate (HR), low frequency/high frequency ratio of HR variability, and baroreflex gain were comparable between control and E-Cul3Δ9 mice, suggesting there was no change in cardiac sympathetic nerve activity. However, low frequency amplitude of arterial pressure variability (16±4 vs 7±2 mmHg 2 ) at night was markedly augmented in E-Cul3Δ9 mice, suggesting increased sympathetic activity in vascular tone regulation. Consistently, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%) and cerebral resistance basilar artery (41% vs 77%). No difference in smooth muscle function was observed. Expression of Cul3Δ9 in primary mouse aortic endothelial cells markedly decreased wild type Cul3 protein, phosphorylated eNOS, and NO production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). Thus, CUL3 mutations in the endothelium contribute to human HT in part through decreased NO bioavailability, endothelial dysfunction and secondary sympathoexcitation.

Cullin-3: Renal and Vascular Mechanisms Regulating Blood Pressure.

The goal of this review is to evaluate recent advances in understanding the pivotal roles of Cullin-3 (CUL3) in blood pressure regulation with a focus on its actions in the kidney and blood vessels. Cul3-based ubiquitin ligase regulates renal electrolyte transport, vascular tone, and redox homeostasis by facilitating the normal turnover of (1) with-no-lysine kinases in the distal nephron, (2) RhoA and phosphodiesterase 5 in the vascular smooth muscle, and (3) nuclear factor E2-related factor 2 in antioxidant responses. CUL3 mutations identified in familial hyperkalemic hypertension (FHHt) yield a mutant protein lacking exon 9 (CUL3∆9) which displays dual gain and loss of function. CUL3∆9 acts in a dominant manner to impair CUL3-mediated substrate ubiquitylation and degradation. The consequent accumulation of substrates and overactivation of downstream signaling cause FHHt through increased sodium reabsorption, enhanced vasoconstriction, and decreased vasodilation. CUL3 ubiquitin ligase maintains normal cardiovascular and renal physiology through posttranslational modification of key substrates which regulate blood pressure. Interference with CUL3 disturbs these key downstream pathways. Further understanding the spatial and temporal specificity of how CUL3 functions in these pathways is necessary to identify novel therapeutic targets for hypertension.

Hypertension-causing cullin 3 mutations disrupt COP9 signalosome binding.

The discovery of new genetic mutations that cause hypertension has illuminated previously unrecognized physiological pathways. One such regulatory pathway was identified when mutations in with no lysine kinase (WNK)4, Kelch-like 3 (KLHL3), and cullin 3 (CUL3) were shown to cause the disease familial hyperkalemic hypertension (FHHt). Mutations in all three genes upregulate the NaCl cotransporter (NCC) due to an impaired ability to degrade WNK protein through the cullin-RING-ligase (CRL) ubiquitin-proteasome system. The CUL3 FHHt mutations cause the most severe phenotype, yet the precise mechanism by which these mutations cause the disease has not been established and current proposed models are controversial. New data have identified a possible novel mechanism involving dysregulation of CUL3 activity by the COP9 signalosome (CSN). The CSN interaction with mutant CUL3 is diminished, causing hyperneddylation of the CRL. Recent work has shown that direct renal CSN impairment mimics some aspects of the CUL3 mutation, including lower KLHL3 abundance and activation of the WNK-NCC pathway. Furthermore, in vitro and in vivo studies of CSN inhibition have shown selective degradation of CRL substrate adaptors via auto-ubiquitination, allowing substrate accumulation. In this review, we will focus on recent research that highlights the role of the CSN role in CUL3 mutations that cause FHHt. We will also highlight how these results inform other recent studies of CSN dysfunction.

Abstract 065: Endothelial CULLIN3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and Hypertension

Mutations in CULLIN3 gene (causing in-frame deletion of exon 9) cause human hypertension (HT), which is likely to be driven by a combination of renal tubular and vascular mechanisms. We have recently shown that smooth muscle expression of Cul3Δ9 mutant causes vascular dysfunction and HT via augmented RhoA/Rho-kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE ERT2 mice. The resultant mice (E-Cul3Δ9) developed nocturnal hypertension (Night time peak systolic BP, E-Cul3Δ9: 138±3 vs Control: 121±4 mmHg, p<0.01) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, p<0.01). No difference was seen in daytime BP. Nitric oxide synthase (NOS) inhibitor L-NAME induced smaller increases in nocturnal peak SBP and DBP in E-Cul3Δ9 mice (15±1 vs 27±3 mmHg, p<0.01), suggesting their NOS activity is low. Of note, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%, p<0.05) and cerebral resistance basilar artery (41% vs 77%, p<0.01). No difference in smooth muscle function was observed. To determine the molecular mechanisms, we isolated primary aortic endothelial cells from mice carrying the inducible Cul3Δ9 construct and induced Cul3Δ9 expression in vitro using adenovirus carrying Cre recombinase gene. Expression of Cul3Δ9 resulted in marked decreases in wild type Cul3 protein, phosphorylated eNOS, and nitric oxide production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). These data define a novel regulatory pathway involving Cullin3/PP2A/phospho-eNOS in the endothelium. Selective endothelial expression of Cul3Δ9 partially phenocopies the hypertension observed in Cul3Δ9 patients, suggesting that mutations in Cullin-3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.

Conditional deletion of smooth muscle Cullin-3 causes severe progressive hypertension.

Patients with mutations in Cullin-3 (CUL3) exhibit severe early onset hypertension but the contribution of the smooth muscle remains unclear. Conditional genetic ablation of CUL3 in vascular smooth muscle (S-CUL3KO) causes progressive impairment in responsiveness to nitric oxide (NO), rapid development of severe hypertension, and increased arterial stiffness. Loss of CUL3 in primary aortic smooth muscle cells or aorta resulted in decreased expression of the NO receptor, soluble guanylate cyclase (sGC), causing a marked reduction in cGMP production and impaired vasodilation to cGMP analogues. Vasodilation responses to a selective large conductance Ca2+-activated K+-channel activator were normal suggesting that downstream signals which promote smooth muscle-dependent relaxation remained intact. We conclude that smooth muscle specific CUL3 ablation impairs both cGMP production and cGMP responses and that loss of CUL3 function selectively in smooth muscle is sufficient to cause severe hypertension by interfering with the NO-sGC-cGMP pathway. Our study provides compelling evidence for the sufficiency of vascular smooth muscle CUL3 as a major regulator of BP. CUL3 mutations cause severe vascular dysfunction, arterial stiffness and hypertension due to defects in vascular smooth muscle.

Abstract 121: Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and Hypertension

Mutations in CULLIN3 gene (causing in-frame deletion of exon 9) cause human hypertension (HT), which is likely to be driven by a combination of renal tubular and vascular mechanisms. We have recently shown that smooth muscle expression of Cul3Δ9 mutant causes vascular dysfunction and HT via augmented RhoA/Rho-kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3Δ9 mice with tamoxifen-inducible Tie2-CRE ERT2 mice. The resultant mice (E-Cul3Δ9) developed nocturnal hypertension (Night time peak systolic BP, E-Cul3Δ9: 138±3 vs Control: 121±4 mmHg, p<0.01) and arterial stiffening (pulse wave velocity, 3.7±0.3 vs 2.7±0.1 m/s, p<0.01). No difference was seen in daytime BP. Nitric oxide synthase (NOS) inhibitor L-NAME induced smaller increases in nocturnal peak SBP and DBP in E-Cul3Δ9 mice (15±1 vs 27±3 mmHg, p<0.01), suggesting their NOS activity is low. Of note, E-Cul3Δ9 mice exhibited impaired endothelial-dependent relaxation in carotid artery (Max ACh relaxation: 69% vs 84%, p<0.05) and cerebral resistance basilar artery (41% vs 77%, p<0.01). No difference in smooth muscle function was observed. To determine the molecular mechanisms, we isolated primary aortic endothelial cells from mice carrying the inducible Cul3Δ9 construct and induced Cul3Δ9 expression in vitro using adenovirus carrying Cre recombinase gene. Expression of Cul3Δ9 resulted in marked decreases in wild type Cul3 protein, phosphorylated eNOS, and nitric oxide production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which dephosphorylates eNOS, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9-induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). These data define a novel regulatory pathway involving Cullin3/PP2A/phospho-eNOS in the endothelium. Selective endothelial expression of Cul3Δ9 partially phenocopies the hypertension observed in Cul3Δ9 patients, suggesting that mutations in Cullin-3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.

Decreased KLHL3 expression is involved in the pathogenesis of pseudohypoaldosteronism type II caused by cullin 3 mutation in vivo.

Pseudohypoaldosteronism type II (PHAII) is a hereditary hypertensive disease caused by mutations in four genes: WNK1, WNK4, Kelch-like3 (KLHL3), and cullin3 (CUL3). Recently, it was revealed that CUL3-KLHL3 E3 ligase complex ubiquitinates WNK1 and WNK4, leading to their degradation, and that a common pathogenesis of PHAII is defective WNK degradation due to CUL3-KLHL3 E3 ligase complex impairment. PHAII-causing CUL3 mutations mediate exon9 skipping, producing a CUL3 protein with a 57-amino acid deletion (Δ403-459). However, the pathogenic effects of KLHL3, an adaptor protein that links WNKs with CUL3, in PHAII caused by CUL3 mutation remain unclear. To clarify detailed pathophysiological mechanisms underlying PHAII caused by CUL3 mutation in vivo, we generated and analyzed knock-in mice carrying the same CUL3 exon9 deletion (CUL3WT/Δex9) as that reported in PHAII patients. CUL3WT/Δex9 mice exhibited a PHAII-like phenotype. Interestingly, we confirmed markedly decreased KLHL3 expression in CUL3WT/Δex9 mice by confirming the true KLHL3 band in vivo. However, the expression of other KLHL family proteins, such as KLHL2, was comparable between WT and mutant mice. KLHL3 expression was decreased in CUL3WT/Δex9 mice. However, expression levels of other KLHL family proteins were comparable between the wild-type and mutant mice. These findings indicate that the decreased abundance of KLHL3 is a specific phenomenon caused by mutant CUL3 (Δexon9). Our findings would improve our understanding of the pathogenesis of PHAII caused by CUL3 mutation in vivo.

Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and Hypertension

Cullin3 (Cul3) is a critical subunit of the Cul3‐Ring‐Ligase (CRL3) ubiquitin ligase complex that targets proteins for proteasomal degradation. Mutations in CULLIN3 gene (causing in‐frame deletion of exon 9, termed Cul3Δ9) cause hypertension in humans. Evidence suggests that the hypertension in these subjects is likely to be caused by a combination of renal tubular and vascular mechanisms. We have recently shown that smooth muscle expression of Cul3Δ9 causes vascular dysfunction and elevation of blood pressure (BP) via augmented RhoA/Rho‐kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo, we bred the conditionally activatable Cul3Δ9 mice with Tek‐CREERT2 mice specifically expressing tamoxifen‐inducible Cre‐recombinase in the endothelium. The resultant mice (E‐Cul3Δ9) exhibited impaired endothelial‐dependent relaxation in carotid artery (maximal relaxation in response to 30 μM acetylcholine, 69% vs 84% in littermate control mice, n=5, p<0.05, two‐way ANOVA repeated measurements) and in the cerebral basilar artery (maximal relaxation in response to 100 μM acetylcholine, 41% vs 77% in littermate control mice, n=5, p<0.01). No difference in smooth muscle function was observed. Moreover, E‐Cul3Δ9 mice exhibited nocturnal hypertension as determined by radiotelemetry (night time peak systolic BP, 138±3 mmHg n=7 vs 122±4 mmHg n=5, p<0.01). However no difference was seen in daytime pressure. Preliminary data indicated that treatment with nitric oxide synthase (NOS) inhibitor Nω‐Nitro‐L‐arginine methyl ester markedly elevated nocturnal peak SBP in control mice (133±5 mmHg, n=3) but not in E‐Cul3Δ9 mice (143±3 mmHg, n=3), suggesting that NOS activity is low in E‐Cul3Δ9 mice. E‐Cul3Δ9 mice also exhibited arterial stiffening as indicated by elevated pulse wave velocity (3.7±0.3 m/s n=5 vs control mice 2.7±0.1 m/s n=7, p<0.01). To determine the molecular mechanism of endothelial dysfunction, primary aortic endothelial cells were isolated from mice carrying the inducible Cul3Δ9 construct and Cul3Δ9 expression was robustly induced by adenovirus carrying Cre recombinase gene in vitro. Expression of Cul3Δ9 resulted in marked decreases in wild type Cul3 protein, phosphorylated eNOS, and nitric oxide production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which negatively regulates eNOS phosphorylation, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9‐induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). These data define a novel regulatory pathway involving Cullin‐3/PP2A/phospho‐eNOS in the endothelium. Selective endothelial expression of Cul3Δ9 partially phenocopies the hypertension observed in Cul3Δ9 patients, suggesting that mutations in Cullin‐3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.Support or Funding InformationDr. Jing Wu is supported by an American Heart Association Postdoctoral Fellowship (17POST33660685). This project is funded by R01 HL125603 and R01 HL131689 to Dr. Curt Sigmund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel mutation in exon 9 of Cullin 3 gene contributes to aberrant splicing in pseudohypoaldosteronism type II.

Pseudohypoaldosteronism type II (PHAII) is a rare renal tubular disease that is inherited in an autosomal dominant manner. Mutations in four genes (WNK1,WNK4,CUL3, and KLHL3) have been identified to be responsible for this disease. Cullin 3 (CUL3) and KLHL3 are subunits of Cullin–RING E3 ubiquitin ligase complexes, and the serine–threonine kinases WNK1 and WNK4 are substrates of this ubiquitin ligase. For CUL3, all mutations associated with PHAII exclusively lead to exon 9 skipping. In this study, we identified a Chinese PHAII kindred caused by a novel synonymous mutation (c.1221A > G p.Glu407Glu) in CUL3, and explored its effects on exon 9 abnormal splicing through an in vitro splicing assay and study of the patients’ RNA. We obtained evidence that this synonymous mutation leads to complete exon 9 skipping, and in silico bioinformatics analysis demonstrated that the CUL3 c.1221A > G mutation might decrease the ratio of exonic splicing enhancers and silencers. This is the first report of PHAII in Chinese patients with a novel CUL3 mutation. Our findings add a novel pathogenic splicing variant to the CUL3 mutational spectrum and provide reference for further research on mechanisms of splicing modulation and development of potential therapeutic reagents for PHAII.

Mechanisms and controversies in mutant Cul3-mediated familial hyperkalemic hypertension.

Autosomal dominant mutations in cullin-3 ( Cul3) cause the most severe form of familial hyperkalemic hypertension (FHHt). Cul3 mutations cause skipping of exon 9, which results in an internal deletion of 57 amino acids from the CUL3 protein (CUL3-∆9). The precise mechanism by which this altered form of CUL3 causes FHHt is controversial. CUL3 is a member of the cullin-RING ubiquitin ligase family that mediates ubiquitination and thus degradation of cellular proteins, including with-no-lysine [K] kinases (WNKs). In CUL3-∆9-mediated FHHt, proteasomal degradation of WNKs is abrogated, leading to overactivation of the WNK targets sterile 20/SPS-1 related proline/alanine-rich kinase and oxidative stress-response kinase-1, which directly phosphorylate and activate the thiazide-sensitive Na+-Cl- cotransporter. Several groups have suggested different mechanisms by which CUL3-∆9 causes FHHt. The majority of these are derived from in vitro data, but recently the Kurz group (Schumacher FR, Siew K, Zhang J, Johnson C, Wood N, Cleary SE, Al Maskari RS, Ferryman JT, Hardege I, Figg NL, Enchev R, Knebel A, O'Shaughnessy KM, Kurz T. EMBO Mol Med 7: 1285-1306, 2015) described the first mouse model of CUL3-∆9-mediated FHHt. Analysis of this model suggested that CUL3-∆9 is degraded in vivo, and thus Cul3 mutations cause FHHt by inducing haploinsufficiency. We recently directly tested this model but found that other dominant effects of CUL3-∆9 must contribute to the development of FHHt. In this review, we focus on our current knowledge of CUL3-∆9 action gained from in vitro and in vivo models that may help unravel this complex problem.

Abstract P189: Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and Hypertension

Mutations in CULLIN3 gene (causing in-frame deletion of exon 9) cause hypertension in humans. The hypertension phenotype is unlikely to be driven by renal tubular mechanisms, as kidney-specific deletion of Cullin3 (Cul3) in mice results in hypotension, not hypertension. We have recently shown that smooth muscle expression of Cul3Δ9 causes vascular dysfunction and elevation of blood pressure (BP) via augmented RhoA/Rho-kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo , we bred the conditionally activatable Cul3α9 mice with Tek-CRE ERT2 mice specifically expressing tamoxifen-inducible Cre-recombinase in the endothelium. The resultant mice (E-Cul3α9) exhibited impaired endothelial-dependent relaxation in the basilar artery (maximal relaxation in response to 30 μM acetylcholine, 45% vs 85% in control mice) and carotid artery. No difference in smooth muscle function was observed. Moreover, E-Cul3α9 mice exhibited nocturnal hypertension as determined by radiotelemetry (night time peak BP, 141±3 mmHg vs 122±3 mmHg). However no difference was seen in daytime pressure. E-Cul3α9 mice also exhibited arterial stiffening as indicated by elevated pulse wave velocity (3.7±0.3 m/s vs 2.7±0.1 m/s). To determine the molecular mechanism of endothelial dysfunction, primary aortic endothelial cells were isolated from mice carrying the inducible Cul3α9 construct and Cul3α9 expression was robustly induced by adenovirus carrying Cre recombinase gene in vitro . Cul3α9 acted in a dominant negative manner by interfering with expression and function of wild type Cul3, leading to impaired turnover of a Cul3 substrate protein phosphatase 2A, marked reduction in phosphorylated eNOS, and decreased nitric oxide production. Treatment with a selective PP2A inhibitor Okadaic Acid (1 nM) rescued Cul3α9-induced impairment of eNOS activity. These data define a novel pathway involving Cullin-3/PP2A/phospho-eNOS in the endothelium. Selective endothelial expression of Cul3α9 partially phenocopies the hypertension observed in Cul3α9 patients, suggesting that mutations in Cullin-3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.

Abstract P243: Smooth Muscle Cullin-3 Deficiency Causes Severe Early Onset Hypertension and Nitric Oxide Resistance

Mutations in Cullin-3 ( Cul3 ) resulting in exon 9 skipping (CUL3α9) cause human hypertension. We demonstrated that selective expression of CUL3α9 in smooth muscle causes arterial stiffness and hypertension. We hypothesized that deletion of CUL3 in smooth muscle causes severe hypertension. Mice carrying a conditional allele of CUL3 were bred with mice expressing a tamoxifen-inducible CRE-recombinase driven by a smooth muscle promoter. Mice were administered tamoxifen i.p. (75 mg/kg) for 5 consecutive days to generate smooth muscle CUL3 knockout (S-CUL3KO). CUL3 protein was undetectable whereas Cullin-1 protein was preserved in aorta from S-CUL3KO mice. We assessed vascular function in the cerebral basilar artery and aorta using pressurized and wire myograph, respectively. Blood pressure (BP) was measured by radiotelemetry. S-CUL3KO mice exhibited significantly increased systolic BP (SBP) at 2 weeks and 4 weeks post tamoxifen compared to corn oil controls (2 wks SBP mmHg: 145±1 vs 115±2, p<0.001; 4 weeks SBP: 169±1 vs 115±3, p<0.001). Pulse wave velocity was also increased in S-CUL3KO mice (3.7±0.1 m/s vs 2.2±0.1, P<0.001), suggesting increased arterial stiffness. Aorta from S-CUL3KO mice exhibited severely impaired vasorelaxation to acetylcholine (ACh) compared to controls (at 100 μM: 1.0±3% vs 77±5%, p<0.0001), and to the nitric oxide donor sodium nitroprusside (SNP) (at 100 μM: 15±4% vs 96±1%, p<0.001). In agreement with data from aorta, cerebral basilar artery from S-CUL3KO mice also exhibited significant impairment to ACh- and SNP-mediated vasorelaxation. Conversely, S-CUL3KO aorta retained an ability to relax in response to a cGMP analogue (8-pCPT-cGMP, at 100 μμ: 8-pCPT-cGMP 70±3%) and to the heme-independent soluble guanylate cyclase activator (BAY 58-2667, at 10 μμ: BAY 58-2667 94±2%), indicating that downstream mechanisms controlled by cGMP remain intact in the absence of CUL3. Captopril (120 mg/kg/day) was sufficient to normalized BP in S-CUL3KO mice to pre-tamoxifen levels (change in SBP mmHg: -50.2±1.9 S-CUL3KO vs -25.0±2 control, P<0.0001). We conclude that smooth muscle CUL3 is a major BP determinant, and identifying novel CUL3 substrates in smooth muscle would be beneficial as therapeutic targets in the treatment of hypertension.

Cullin-3 mutation causes arterial stiffness and hypertension through a vascular smooth muscle mechanism.

Cullin-3 (CUL3) mutations (CUL3Δ9) were previously identified in hypertensive patients with pseudohypoaldosteronism type-II (PHAII), but the mechanism causing hypertension and whether this is driven by renal tubular or extratubular mechanisms remains unknown. We report that selective expression of CUL3Δ9 in smooth muscle acts by interfering with expression and function of endogenous CUL3, resulting in impaired turnover of the CUL3 substrate RhoA, increased RhoA activity, and augmented RhoA/Rho kinase signaling. This caused vascular dysfunction and increased arterial pressure under baseline conditions and a marked increase in arterial pressure, collagen deposition, and vascular stiffness in response to a subpressor dose of angiotensin II, which did not cause hypertension in control mice. Inhibition of total cullin activity increased the level of CUL3 substrates cyclin E and RhoA, and expression of CUL3Δ9 decreased the level of the active form of endogenous CUL3 in human aortic smooth muscle cells. These data indicate that selective expression of the Cul3Δ9 mutation in vascular smooth muscle phenocopies the hypertension observed in Cul3Δ9 human subjects and suggest that mutations in CUL3 cause human hypertension in part through a mechanism involving smooth muscle dysfunction initiated by a loss of CUL3-mediated degradation of RhoA.

Abstract P158: Cullin3 Regulated Endothelial Function by Modulating eNOS Activity

Pseudohypoaldosteronism type II (PHAII) patients expressing dominant negative cullin3 mutations exhibit increased renal NaCl reabsorption and develop hyperkalaemia, metabolic acidosis and hypertension. It is unclear whether loss of cullin3 function in extra-renal tissues contributes to the hypertensive phenotype. In the vasculature, endothelial Nrf2 stability is tightly regulated by cullin3-based E3 ubiquitin ligase via the redox-sensitive adaptor kelch-like ECH-associated protein 1. In the present study, we found that 24-hour treatment with a pan cullin inhibitor MLN4924 (1 μM) caused a 3-fold increase in Nrf2 protein in mouse lung endothelial cells (MLECs), while tert-butyl hydroperoxide (tBHP, 240 μM) had no effect on Nrf2 level. However, both MLN4924 and tBHP triggered time-dependent accumulation of Nrf2 in the nuclei, which peaked at 40 minutes following treatment. As a result, both treatments induced marked upregulation of antioxidant genes including NAD(P)H quinone oxidoreductase 1, heme oxygenase 1, glutamate cysteine ligase (rate-limiting enzyme in glutathione synthesis), and catalase both in MLECs and primary mouse aortic endothelial cells (MAECs). Of note, MLN4924 upregulated Nox4 expression (1.0 ± 0.15 vs 1.7 ± 0.2) and tBHP upregulated Nox1 (1.0 ± 0.2 vs 4.8 ± 1.1), while MLN4924 and tBHP both markedly increased intracellular superoxide as determined by dihydroethidium staining. In addition, intracellular nitric oxide was decreased by half in MLN4924-treated MLECs. This redox imbalance was likely due to impaired eNOS expression and activation as MLN4924 caused a 25% reduction in total eNOS and a 75% reduction in phosphorylated eNOS, while tBHP lead to a 50% reduction in phosphorylated eNOS with no effect on total eNOS. This suggests that decreased eNOS activation contributed to the oxidative stress induced by these agents. These data imply that suppression of cullin3 in arterial endothelial cells may dampen endothelium-dependent vascular relaxation and contribute to the blood pressure elevation observed in PHAII patients with global loss of cullin3 function. Although cullin3 also negatively regulates Nrf2-mediated antioxidant responses in vascular endothelial cells, this likely occurs as a compensatory mechanism.

ROMK expression remains unaltered in a mouse model of familial hyperkalemic hypertension caused by the CUL3Δ403-459 mutation.

Familial hyperkalemic hypertension (FHHt) is a rare inherited form of salt‐dependent hypertension caused by mutations in proteins that regulate the renal Na+‐Cl‐ cotransporter NCC. Mutations in four genes have been reported to cause FHHt including CUL3 (Cullin3) that encodes a component of a RING E3 ligase. Cullin‐3 binds to WNK kinase‐bound KLHL3 (the substrate recognition subunit of the ubiquitin ligase complex) to promote ubiquitination and proteasomal degradation of WNK kinases. Deletion of exon 9 from CUL3 (affecting residues 403‐459, CUL3Δ403‐459) causes a severe form of FHHt (PHA2E) that is recapitulated closely in a knock‐in mouse model. The loss of functionality of CUL3Δ403‐459 and secondary accumulation of WNK kinases causes substantial NCC activation. This accounts for the hypertension in FHHt but the origin of the hyperkalemia is less clear. Hence, we explored the impact of CUL3Δ403‐459 on expression of the distal secretory K channel, ROMK, both in vitro and in vivo. We found that expressing wild‐type but not the CUL3Δ403‐459 mutant form of CUL3 prevented the suppression of ROMK currents by WNK4 expressed in Xenopus oocytes. The mutant CUL3 protein was also unable to affect ROMK‐EGFP protein expression at the surface of mouse M‐1 cortical collecting duct (CCD) cells. The effects of CUL3 on ROMK expression in both oocytes and M‐1 CCD cells was reduced by addition of the neddylation inhibitor, MLN4924. This confirms that neddylation is important for CUL3 activity. Nevertheless, in our knock‐in mouse model expressing CUL3Δ403‐459 we could not show any alteration in ROMK expression by either western blotting whole kidney lysates or confocal microscopy of kidney sections. This suggests that the hyperkalemia in our knock‐in mouse and human PHA2E subjects with the CUL3Δ403‐459 mutation is not caused by reduced ROMK expression in the distal nephron.

Degradation by Cullin 3 and effect on WNK kinases suggest a role of KLHL2 in the pathogenesis of Familial Hyperkalemic Hypertension

Mutations in WNK1 and WNK4, and in components of the Cullin-Ring Ligase system, kelch-like 3 (KLHL3) and Cullin 3 (CUL3), can cause the rare hereditary disease, Familial Hyperkalemic Hypertension (FHHt). The disease is characterized by overactivity of the renal sodium chloride cotransporter (NCC), which is phosphorylated and activated by the WNK-stimulated Ste20-type kinases, SPAK and OSR1. WNK kinases themselves can be targeted for ubiquitination and degradataion by the CUL3-KLHL3 E3 ubiquitin ligase complex. It is unclear, however, why there are significant differences in phenotypic severity among FHHt patients with mutations in different genes. It was reported that kelch-like 2 (KLHL2), a homolog of KLHL3, can also target WNK kinases for ubiquitation and degradation, and may play a special role in the systemic vasculature. Our recent study revealed the disease mutant CUL3 exhibits enhanced degradation of its adaptor protein KLHL3, potentially resulting in accumulation of WNK kinases secondarily. To investigate if KLHL2 plays a role in FHHt, we studied the effect of wild type and FHHt mutant CUL3 on degradation of KLHL2 and WNK kinase proteins in HEK293 cells. Although CUL3 facilitates KLHL2 degradation, the disease mutant CUL3 is more active in this regard. KLHL2 facilitated the degradation of wild type but not disease mutant WNK4 protein. These results suggest that KLHL2 likely plays a role in the pathogenesis of FHHt, and aggravates the phenotype caused by mutations in CUL3 and WNK4.

Hypertension-causing Mutations in Cullin3 Protein Impair RhoA Protein Ubiquitination and Augment the Association with Substrate Adaptors

Cullin-Ring ubiquitin ligases regulate protein turnover by promoting the ubiquitination of substrate proteins, targeting them for proteasomal degradation. It has been shown previously that mutations in Cullin3 (Cul3) causing deletion of 57 amino acids encoded by exon 9 (Cul3Δ9) cause hypertension. Moreover, RhoA activity contributes to vascular constriction and hypertension. We show that ubiquitination and degradation of RhoA is dependent on Cul3 in HEK293T cells in which Cul3 expression is ablated by either siRNA or by CRISPR-Cas9 genome editing. The latter was used to generate a Cul3-null cell line (HEK293T(Cul3KO)). When expressed in these cells, Cul3Δ9 supported reduced ubiquitin ligase activity toward RhoA compared with equivalent levels of wild-type Cul3 (Cul3WT). Consistent with its reduced activity, binding of Cul3Δ9 to the E3 ubiquitin ligase Rbx1 and neddylation of Cul3Δ9 were impaired significantly compared with Cul3WT. Conversely, Cul3Δ9 bound to substrate adaptor proteins more efficiently than Cul3WT. Cul3Δ9 also forms unstable dimers with Cul3WT, disrupting dimers of Cul3WT complexes that are required for efficient ubiquitination of some substrates. Indeed, coexpression of Cul3WT and Cul3Δ9 in HEK293T(Cul3KO) cells resulted in a decrease in the active form of Cul3WT. We conclude that Cul3Δ9-associated ubiquitin ligase activity toward RhoA is impaired and suggest that Cul3Δ9 mutations may act dominantly by sequestering substrate adaptors and disrupting Cul3WT complexes.