Kelch-Like Protein 2 Mediates Angiotensin II–With No Lysine 3 Signaling in the Regulation of Vascular Tonus

Abstract

Recently, the kelch-like protein 3 (KLHL3)-Cullin3 complex was identified as an E3 ubiquitin ligase for with no lysine (WNK) kinases, and the impaired ubiquitination of WNK4 causes pseudohypoaldosteronism type II (PHAII), a hereditary hypertensive disease. However, the involvement of WNK kinase regulation by ubiquitination in situations other than PHAII has not been identified. Previously, we identified the WNK3-STE20/SPS1-related proline/alanine-rich kinase-Na/K/Cl cotransporter isoform 1 phosphorylation cascade in vascular smooth muscle cells and found that it constitutes an important mechanism of vascular constriction by angiotensin II (AngII). In this study, we investigated the involvement of KLHL proteins in AngII-induced WNK3 activation of vascular smooth muscle cells. In the mouse aorta and mouse vascular smooth muscle (MOVAS) cells, KLHL3 was not expressed, but KLHL2, the closest homolog of KLHL3, was expressed. Salt depletion and acute infusion of AngII decreased KLHL2 and increased WNK3 levels in the mouse aorta. Notably, the AngII-induced changes in KLHL2 and WNK3 expression occurred within minutes in MOVAS cells. Results of KLHL2 overexpression and knockdown experiments in MOVAS cells confirmed that KLHL2 is the major regulator of WNK3 protein abundance. The AngII-induced decrease in KLHL2 was not caused by decreased transcription but increased autophagy-mediated degradation. Furthermore, knockdown of sequestosome 1/p62 prevented the decrease in KLHL2, suggesting that the mechanism of KLHL2 autophagy could be selective autophagy mediated by sequestosome 1/p62. Thus, we identified a novel component of signal transduction in AngII-induced vascular contraction that could be a promising drug target. 26: 212926: -213826: , 201526: . doi: 10.1681 Recently, the kelch-like protein 3 (KLHL3) and Cullin3 (Cul3) were identified as the genes responsible for a hereditary hypertensive diseasepseudohypoaldosteronism type II (PHAII). 1 Because the with no lysine (WNK) kinases (WNK1 and WNK4) had already been identified as the responsible genes for PHAII and the KLHL proteins were known to serve as substrate adaptors of Cul3-based E3 ubiquitin ligase, 1-3 we speculated and recently showed that KLHL3 functions as an E3 ligase with Cul3 for WNK4 and that the impaired ubiquitination of WNK4 and its subsequent increase within the cell stimulates the downstream OSR1/STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NaCl cotransporter signaling and causes PHAII. 4 In addition to WNK4, WNK1 and other WNK kinases (WNK2 and WNK3) have been identified as substrates of KLHL3-Cul3 E3 ligase, because KLHL3 can bind to all WNKs in a highly conserved domain (acidic domain). 5 Furthermore, we recently reported that KLHL2 possesses a kelch repeat domain (WNK binding domain) highly similar to that of KLHL3 and that J Am Soc Nephrol

Loop diuretics affect skeletal myoblast differentiation and exercise-induced muscle hypertrophy

“…We speculated that the inhibition of NKCC1-mediated GCl affects Ca 2+ transients following membrane depolarization in skeletal muscle cells similarly as seen in vascular smooth muscle cells1718. Thus, we analyzed the effect of bumetanide and furosemide on depolarization-evoked calcium transients after KCl treatment.…”

KLHL3 Knockout Mice Reveal the Physiological Role of KLHL3 and the Pathophysiology of Pseudohypoaldosteronism Type II Caused by Mutant KLHL3

“…Recent studies have focused on WNK signaling in extrarenal tissue and have found that WNK signaling also plays a role in other tissues, including vascular smooth muscle cells (9,26,38), the brain (39-41), neurons (42), the intestine (43), and the heart (44). However, the tissue distribution and intratissue localization of KLHL3 remained unclear.…”

“…Similar to KLHL3, KLHL2 directly interacts with WNK kinases, leading to their ubiquitination and degradation (45,46). Zeniya et al recently reported that ubiquitination by KLHL2 is the major regulator of WNK3 protein levels in response to angiotensin II stimulation, although other in vivo roles of KLHL2 were not determined (38).…”

Multistep regulation of autophagy by WNK1

“…WNKs are also regulated by binding Kelch-like adaptors, KLHL2 and KLHL3, which target them for degradation (12)(13)(14)(15)(16). Concentrations of WNKs can be changed relatively rapidly in cells by this type of degradative process with pathophysiological implications (70). Changing the WNK1 amount through degradation, distinct from changing WNK1 activity, may be a physiologically significant mechanism to induce autophagy.…”