Lactate programs PBX1 lactylation and mesangial proliferation in lupus nephritis
Enzhuo Liu, Chenghua Weng, Chenchu Yan, Xingchen Zhu, Xinyue Li, Mengdi Liu, Zhenke Wen, Zhichun Liu
Enzhuo Liu, Chenghua Weng, Chenchu Yan, Xingchen Zhu, Xinyue Li, Mengdi Liu, Zhenke Wen, Zhichun Liu
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Research Article Metabolism

Lactate programs PBX1 lactylation and mesangial proliferation in lupus nephritis

Abstract

Lupus nephritis (LN) constitutes the most common organ-threatening manifestation of systemic lupus erythematosus (SLE), with the pathological proliferation of mesangial cells (MCs) recognized as a critical factor in its pathogenesis and progression. Self-DNA-containing immune complex (DNA-IC) represents a prime pathogenic factor in SLE, yet its pathological effect on MCs remains unclear. In the present study, we elucidated the mechanism underlying the excessive proliferation of MCs following the recognition of DNA-IC in patients with LN. Here, we pinpointed that the excessive proliferation of MCs was attributed to an anomalous transition from the G1 to the S phase of the cell cycle in patients with LN. Mechanically, the dysfunction of P27 protein resulted in the aberrant G1-S phase transition, and the phenomenon was closely related to the ubiquitin-mediated degradation of its key transcription factor, PBX1. This degradation was regulated by lactylation of PBX1 in the site of Lys40 residue. The elevated lactylation level of PBX1 protein was caused by the upregulation of glycolysis levels induced by DNA-IC. Accordingly, targeting lactate production in MCs from patients with LN effectively alleviated their renal inflammation and fibrosis progression. Elevated lactate resulted in PBX1 lactylation, leading to excessive proliferation of MCs and, thus, serving as a promising therapeutic target for LN.

Authors

Enzhuo Liu, Chenghua Weng, Chenchu Yan, Xingchen Zhu, Xinyue Li, Mengdi Liu, Zhenke Wen, Zhichun Liu

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Figure 3

Ubiquitination of PBX1 accounts for its deficiency in MCs.

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Ubiquitination of PBX1 accounts for its deficiency in MCs. (A) The endog...
(A) The endogenous immunoprecipitated PBX1 protein from LN and HC MCs was detected in order to analyze the level of ubiquitination. IP: PBX1 were analyzed on a different gel using the same biological samples; input: PBX1 and β-actin were detected on a separate gel from the same samples (n = 7). (B) HEK293 cells were transfected with different types of HA-Ub plasmids and stimulated with the presence or absence of DNA-IC. Flag-PBX1 was immunoprecipitated for detection of ubiquitination. IP: Flag was analyzed on a different gel using the same biological samples; input: Flag and β-actin were detected on a separate gel from the same samples. (C) Mass spectrometry was conducted to screen for possible E3 ligases that bind with PBX1 in LN MCs. E3 ubiquitin ligases were screened from both the HC and LN groups, and only proteins with more than 1 peptide are shown. Furthermore, RT-qPCR was used to analyze the relative expression of ligase mRNA, which was compared in order to define candidate E3 ubiquitin ligase. Data are from 6 independent experiments. (D and E) TRIM21 shRNAs were transfected into MCs, and immunoblotting was used to detect PBX1 protein level and the ubiquitination level of endogenous immunoprecipitated PBX1 protein (in D, lanes were run from the same sample on different gels using immunoblot and immunoprecipitation; in E, lanes were run on the same gel). (F) TRIM21 mRNA expression was compared between LN and HC MCs using RT-qPCR in 6 independent experiments. (G) Immunoblot analysis of TRIM21 protein in LN and HC MCs. Lanes were run on the same gel (n = 4). (H) The binding of PBX1 to TRIM21 in LN and HC MCs was detected by coimmunoprecipitation. Lanes were run from the same sample from different gel using immunoblot and immunoprecipitation. Lines with whiskers show the mean ± SEM. *P < 0.05, ***P < 0.001 with ANOVA plus Turkey’s method (C) and paired t test (A and F–H).