Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia

Deletion of exon 9 from Cullin‐3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds the RING protein RBX1 and various substrate adaptors to form Cullin‐RING‐ubiquitin‐ligase complexes. Bound to KLHL3, CUL3‐RBX1 ubiquitylates WNK kinases, promoting their ubiquitin‐mediated proteasomal degradation. Since WNK kinases activate Na/Cl co‐transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin‐RING‐ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto‐ubiquitylates and loses interaction with two important Cullin regulators: the COP9‐signalosome and CAND1. A novel knock‐in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases

Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia

This is the final version of the article. Available from the publisher via the DOI in this record.Deletion of exon 9 from Cullin‐3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds the RING protein RBX1 and various substrate adaptors to form Cullin‐RING‐ubiquitin‐ligase complexes. Bound to KLHL3, CUL3‐RBX1 ubiquitylates WNK kinases, promoting their ubiquitin‐mediated proteasomal degradation. Since WNK kinases activate Na/Cl co‐transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin‐RING‐ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto‐ubiquitylates and loses interaction with two important Cullin regulators: the COP9‐signalosome and CAND1. A novel knock‐in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases.This work was supported by the British Heart Foundation (a PhD studentship to KS and PG 13 89 30577), Medical Research Council, and an ERC Starting Investigator Grant (to TK), as well as the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Merck, Janssen Pharmaceutica and Pfizer). The Human Research Tissue Bank is supported by the NIHR Cambridge Biomedical Research Centre

Genome-wide association study identifies six new loci influencing pulse pressure and mean arterial pressure.

Numerous genetic loci have been associated with systolic blood pressure (SBP) and diastolic blood pressure (DBP) in Europeans. We now report genome-wide association studies of pulse pressure (PP) and mean arterial pressure (MAP). In discovery (N = 74,064) and follow-up studies (N = 48,607), we identified at genome-wide significance (P = 2.7 × 10(-8) to P = 2.3 × 10(-13)) four new PP loci (at 4q12 near CHIC2, 7q22.3 near PIK3CG, 8q24.12 in NOV and 11q24.3 near ADAMTS8), two new MAP loci (3p21.31 in MAP4 and 10q25.3 near ADRB1) and one locus associated with both of these traits (2q24.3 near FIGN) that has also recently been associated with SBP in east Asians. For three of the new PP loci, the estimated effect for SBP was opposite of that for DBP, in contrast to the majority of common SBP- and DBP-associated variants, which show concordant effects on both traits. These findings suggest new genetic pathways underlying blood pressure variation, some of which may differentially influence SBP and DBP

Functional characterization of common BCL11B gene desert variants suggests a lymphocyte-mediated association of BCL11B with aortic stiffness.

The recent genome-wide analysis of carotid-femoral pulse wave velocity (PWV) identified a significant locus within the 14q32.2 gene desert. Gene regulatory elements for the transcriptional regulator B-cell CLL/lymphoma 11B (BCL11B) are within this locus and an attractive target for the gene association. We investigated the functional impact of these gene desert SNPs on BCL11B transcript in human aorta to characterize further its role in aortic stiffness. To do this, we used a large repository of aortic tissues (n = 185) from an organ transplant program and assessed ex vivo stiffness of the aortic rings. We tested association of three lead SNPs from the GWAS meta-analysis with ex vivo aortic stiffness and BCL11B aortic mRNA expression: rs1381289 and rs10782490 SNPs associated significantly with PWV and showed allele-specific differences in BCL11B mRNA. The risk alleles associated with lower BCL11B expression, suggesting a protective role for BCL11B. Despite strong association, we could not detect BCL11B protein in the human aorta. However, qPCR for CD markers showed that BCL11B transcript correlated strongly with markers for activated lymphocytes. Our data confirm the significance of the 14q32.2 region as a risk locus for aortic stiffness and an upstream regulator of BCL11B. The BCL11B transcript detected in the human aorta may reflect lymphocyte infiltration, suggesting that immune mechanisms contribute to the observed association of BCL11B with aortic stiffness.This work was funded by a PhD studentship for RAM from the Omani Government, Rosetrees Trust (Ref. CM374), British Heart Foundation (FS12/8/29377) and the UK NIHR

Functional characterization of common BCL11B gene desert variants suggests a lymphocyte-mediated association of BCL11B with aortic stiffness.

The recent genome-wide analysis of carotid-femoral pulse wave velocity (PWV) identified a significant locus within the 14q32.2 gene desert. Gene regulatory elements for the transcriptional regulator B-cell CLL/lymphoma 11B (BCL11B) are within this locus and an attractive target for the gene association. We investigated the functional impact of these gene desert SNPs on BCL11B transcript in human aorta to characterize further its role in aortic stiffness. To do this, we used a large repository of aortic tissues (n = 185) from an organ transplant program and assessed ex vivo stiffness of the aortic rings. We tested association of three lead SNPs from the GWAS meta-analysis with ex vivo aortic stiffness and BCL11B aortic mRNA expression: rs1381289 and rs10782490 SNPs associated significantly with PWV and showed allele-specific differences in BCL11B mRNA. The risk alleles associated with lower BCL11B expression, suggesting a protective role for BCL11B. Despite strong association, we could not detect BCL11B protein in the human aorta. However, qPCR for CD markers showed that BCL11B transcript correlated strongly with markers for activated lymphocytes. Our data confirm the significance of the 14q32.2 region as a risk locus for aortic stiffness and an upstream regulator of BCL11B. The BCL11B transcript detected in the human aorta may reflect lymphocyte infiltration, suggesting that immune mechanisms contribute to the observed association of BCL11B with aortic stiffness.This work was funded by a PhD studentship for RAM from the Omani Government, Rosetrees Trust (Ref. CM374), British Heart Foundation (FS12/8/29377) and the UK NIHR

Oxidative DNA damage promotes vascular aging associated with changes in extracellular matrix-regulating proteins.

AIMS: Vascular aging is characterized by vessel stiffening, with increased deposition of extracellular matrix (ECM) proteins including collagens. Oxidative DNA damage occurs in vascular aging, but how it regulates ECM proteins and vascular stiffening is unknown. We sought to determine the relationship between oxidative DNA damage and ECM regulatory proteins in vascular aging. METHODS AND RESULTS: We examined oxidative DNA damage, the major base excision repair (BER) enzyme 8-Oxoguanine DNA Glycosylase (Ogg1) and its regulators, multiple physiological markers of aging, and ECM proteomics in mice from 22-72w. Vascular aging was associated with increased oxidative DNA damage, and decreased expression of Ogg1, its active acetylated form, its acetylation regulatory proteins P300 and CBP, and the transcription factor Foxo3a. Vascular stiffness was examined in vivo in control, Ogg1-/-, or mice with vascular smooth muscle cell-specific expression of Ogg1+ (Ogg1) or an inactive mutation (Ogg1KR). Ogg1-/- and Ogg1KR mice showed reduced arterial compliance and distensibility, and increased stiffness and pulse pressure, whereas Ogg1 expression normalised all parameters to 72w. ECM proteomics identified major changes in collagens with aging, and downregulation of the ECM regulatory proteins Protein 6-lysyl oxidase (LOX) and WNT1-inducible-signaling pathway protein 2 (WISP2). Ogg1 overexpression upregulated LOX and WISP2 both in vitro and in vivo, and downregulated Transforming growth factor β1 (TGFb1) and Collagen 4α1 in vivo compared with Ogg1KR. Foxo3a activation induced Lox, while Wnt3 induction of Wisp2 also upregulated LOX and Foxo3a, and downregulated TGFβ1 and fibronectin 1. In humans, 8-oxo-G increased with vascular stiffness, while active OGG1 reduced with both age and stiffness. CONCLUSIONS: Vascular aging is associated with oxidative DNA damage, downregulation of major BER proteins, and changes in multiple ECM structural and regulatory proteins. Ogg1 protects against vascular aging, associated with changes in ECM regulatory proteins including LOX and WISP2

The matrix proteins aggrecan and fibulin-1 play a key role in determining aortic stiffness

Stiffening of the aorta is an important independent risk factor for myocardial infarction and stroke. Yet its genetics is complex and little is known about its molecular drivers. We have identified for the first time, tagSNPs in the genes for extracellular matrix proteins, aggrecan and fibulin-1, that modulate stiffness in young healthy adults. We confirmed SNP associations with ex vivo stiffness measurements and expression studies in human donor aortic tissues. Both aggrecan and fibulin-1 were found in the aortic wall, but with marked differences in the distribution and glycosylation of aggrecan reflecting loss of chondroitin-sulphate binding domains. These differences were age-dependent but the striking finding was the acceleration of this process in stiff versus elastic young aortas. These findings suggest that aggrecan and fibulin-1 have critical roles in determining the biomechanics of the aorta and their modification with age could underpin age-related aortic stiffening

Oxidative DNA damage promotes vascular aging associated with changes in extracellular matrix-regulating proteins

AIMS: Vascular aging is characterized by vessel stiffening, with increased deposition of extracellular matrix (ECM) proteins including collagens. Oxidative DNA damage occurs in vascular aging, but how it regulates ECM proteins and vascular stiffening is unknown. We sought to determine the relationship between oxidative DNA damage and ECM regulatory proteins in vascular aging.METHODS AND RESULTS: We examined oxidative DNA damage, the major base excision repair (BER) enzyme 8-Oxoguanine DNA Glycosylase (Ogg1) and its regulators, multiple physiological markers of aging, and ECM proteomics in mice from 22-72w. Vascular aging was associated with increased oxidative DNA damage, and decreased expression of Ogg1, its active acetylated form, its acetylation regulatory proteins P300 and CBP, and the transcription factor Foxo3a. Vascular stiffness was examined in vivo in control, Ogg1-/-, or mice with vascular smooth muscle cell-specific expression of Ogg1+ (Ogg1) or an inactive mutation (Ogg1KR). Ogg1-/- and Ogg1KR mice showed reduced arterial compliance and distensibility, and increased stiffness and pulse pressure, whereas Ogg1 expression normalised all parameters to 72w. ECM proteomics identified major changes in collagens with aging, and downregulation of the ECM regulatory proteins Protein 6-lysyl oxidase (LOX) and WNT1-inducible-signaling pathway protein 2 (WISP2). Ogg1 overexpression upregulated LOX and WISP2 both in vitro and in vivo, and downregulated Transforming growth factor β1 (TGFb1) and Collagen 4α1 in vivo compared with Ogg1KR. Foxo3a activation induced Lox, while Wnt3 induction of Wisp2 also upregulated LOX and Foxo3a, and downregulated TGFβ1 and fibronectin 1. In humans, 8-oxo-G increased with vascular stiffness, while active OGG1 reduced with both age and stiffness.CONCLUSIONS: Vascular aging is associated with oxidative DNA damage, downregulation of major BER proteins, and changes in multiple ECM structural and regulatory proteins. Ogg1 protects against vascular aging, associated with changes in ECM regulatory proteins including LOX and WISP2.</p