Department of Immunology, and Key Laboratory of Immunological Environment and Disease, Nanjing Medical University, Nanjing, China.
Department of Central Laboratory, The Affiliated Huaian No. 1 People's Hospital, Nanjing Medical University, Huai'an, China.
Int Immunopharmacol. 2023 Nov;124(Pt B):110970. doi: 10.1016/j.intimp.2023.110970. Epub 2023 Sep 23.
Rat Thy-1 nephritis (Thy-1N) is an experimental model for studying human mesangioproliferative glomerulonephritis (MsPGN), and its pathological features are glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) accumulation. Although we have confirmed that renal lesions of Thy-1N rats are sublytic C5b-9-dependent, and ECM accumulation is related to tissue inhibitor of matrix metalloproteinase (TIMP) inhibiting matrix metalloproteinase (MMP) activity, whether sublytic C5b-9 can induce TIMP production by GMC in Thy-1N rat and the underlying mechanism remains unclear. In the study, we proved that the expressions of TIMP3, krϋppel-like transcription factor 5 (KLF5) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were simultaneously up-regulated both in the renal tissues of Thy-1N rats (in vivo) and in the GMC exposed to sublytic C5b-9 (in vitro). Further mechanism exploration discovered that KLF5 and TRAF6 as two upstream molecules could induce TIMP3 gene transcription through binding to the same region i.e., -1801nt to -1554nt (GGGGAGGGGC) and -228nt to -46nt (GCCCCGCCCC) of TIMP3 promoter. In the process, TRAF6 mediated KLF5 K63-linked ubiquitination at K99 and K100 enhancing KLF5 nuclear localization and binding to TIMP3 promoter, augmenting its gene activation. Furthermore, the experiments in vivo exhibited that silencing KLF5, TRAF6 or TIMP3 gene could markedly lessen renal KLF5 K63-linked ubiquitination or TIMP3 induction, ECM accumulation and other pathological changes of Thy-1N rats. Besides, the positive expressions of above-mentioned these proteins and ECM accumulation and their correlation in the renal tissues of MsPGN patients were also demonstrated. Overall, our findings implicate that KLF5 and TRAF6 play a promoting role in sublytic C5b-9-triggered TIMP3 gene transcription and expression, which might provide a novel mechanistic insight into rat Thy-1N and human MsPGN.
大鼠 Thy-1 肾炎（Thy-1N）是研究人类系膜增生性肾小球肾炎（MsPGN）的实验模型，其病理特征为肾小球系膜细胞（GMC）增殖和细胞外基质（ECM）积聚。虽然我们已经证实，Thy-1N 大鼠的肾脏病变是亚致死性 C5b-9 依赖性的，ECM 积聚与组织抑制剂金属蛋白酶（TIMP）抑制基质金属蛋白酶（MMP）活性有关，但亚致死性 C5b-9 是否能诱导 Thy-1N 大鼠 GMC 产生 TIMP 以及潜在机制尚不清楚。在本研究中，我们证明了 TIMP3、Krüppel 样转录因子 5（KLF5）和肿瘤坏死因子受体相关因子 6（TRAF6）的表达在 Thy-1N 大鼠的肾脏组织中（体内）和亚致死性 C5b-9 暴露的 GMC 中（体外）同时上调。进一步的机制探索发现，KLF5 和 TRAF6 作为两个上游分子，可通过结合 TIMP3 启动子的相同区域（-1801nt 至-1554nt[GGGGAGGGGC]和-228nt 至-46nt[GCCCCGCCCC]）诱导 TIMP3 基因转录。在这个过程中，TRAF6 介导 KLF5 K63 连接的泛素化，在 K99 和 K100 上修饰 KLF5，促进其核定位并与 TIMP3 启动子结合，增强其基因激活。此外，体内实验表明，沉默 KLF5、TRAF6 或 TIMP3 基因可显著减少 Thy-1N 大鼠肾脏中 KLF5 K63 连接的泛素化或 TIMP3 的诱导、ECM 积聚和其他病理变化。此外，还在 MsPGN 患者的肾脏组织中证实了上述这些蛋白的阳性表达、ECM 积聚及其相关性。总的来说，我们的研究结果表明，KLF5 和 TRAF6 在亚致死性 C5b-9 触发的 TIMP3 基因转录和表达中起促进作用，这可能为大鼠 Thy-1N 和人类 MsPGN 提供新的机制见解。
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