LRRC8A Anion Channel Knockout Stimulates the Nrf2 Antioxidant Response

Abstract

Leucine Rich Repeat Containing 8A (LRRC8A) is an essential component of volume‐regulated anion channels (VRACs), which regulate cell volume. These anion channels are activated by pro-inflammatory mediators such as angiotensin II and tumor necrosis factor-α (TNFα). We previously demonstrated that VRAC inhibition or LRRC8A knockdown reduced Nox1 activation and impaired the inflammatory response to TNFα in cultured vascular smooth muscle cells. We hypothesized that loss of LRRC8A would also modify the antioxidant status of VSMCs. Vascular smooth muscle-specific (Sm22alpha-Cre) LRRC8A KO mice were created. Primary VSMC cultures were prepared from aortae and mesenteric arteries from male mice. Western blotting of protein from WT and LRRC8A KO mesenteric artery VSMCs revealed significant upregulation of proteins that are known to be under the control of the Nrf-2 antioxidant response; heme oxygenase 1 (2.1 fold), thioredoxin 1 (1.8 fold), and superoxide dismutase 1 (1.3 fold) superoxide dismutase 3 (1.8 fold). There was no change in the abundance of catalase.To further explore the role of LRRC8A in intracellular signaling, LRRC8A was immunoprecipitated from cultured WT and LRRC8A KO VSMCs and proteins were analyzed by Mass Spectrometry (MS). Reads were sorted for proteins identified in wild type but not in KO cells. This approach identified the Trim21 E3 ubiquitin ligase, a known regulator of the antioxidant response, as an LRRC8A-associated protein. Trim21 ubiquitinates and thereby promotes degradation of p62 (Sequestosome-1, SQSTM1). p62 sequesters Kelch-like ECH-associated protein 1 (Keap1), preventing it from promoting ubiquitination and destruction of Nrf-2 by the proteosome. Consistent with this, TRIM21 KO mice have an augmented antioxidant status and are protected from myocardial inflammation in response to doxorubicin or ligation of the left coronary artery. We considered the possibility that association of TRIM21 with LRRC8A (directly or indirectly) regulates its activity. Co-immunoprecipitation and western blotting confirmed association of LRRC8A with TRIM21. Pulldown of ubiquitinated proteins from WT and KO VSMCs failed to provide evidence that LRRC8A is ubiquitinated, however, p62 ubiquitination was markedly reduced in KO VSMCs. Thus, reduced p62 turnover in LRRC8A KO cells may enhance Keap1 sequestration and increase Nrf2 activity. The ability of LRRC8A knockdown to both impair the inflammatory response to TNFα and to enhance the antioxidant status of VSMCs identify this protein as an appealing target for the treatment of vascular inflammation. This work was supported by our both grant from NIDDK (1R01DK132948) and NIH (1 R01 HL160975-01A1). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.