IL-33 has emerged as a central mediator of immune, inflammatory, and fibrotic responses. Many studies have focused on mature IL-33, but elevated expression of the precursor, full-length IL-33 (FLIL33), has also been implicated in a spectrum of diseases, including tissue fibrosis. We previously reported and now confirmed that overexpression of FLIL33 induced phosphorylation of the key profibrotic signaling mediator of TGF-β, Smad3, in primary human lung fibroblasts from healthy donors and idiopathic pulmonary fibrosis patients. Presently, we demonstrate that FLIL33-induced Smad3 phosphorylation was not abrogated by anti-TGF-β antibody but was abrogated by ALK5/TGFBR1-specific and Smad3-specific inhibition, indicating that FLIL33 effect was independent of TGF-β but dependent on its receptor, TGFBR. Western blotting analyses revealed that FLIL33 overexpression increased levels, but did not affect subcellular distribution, of the AP2A1 and AP2B1 subunits of the adaptor protein complex 2 (AP2), a known TGFBR binding partner. siRNA-mediated inhibition of these subunits blocked FLIL33-induced Smad3 phosphorylation, whereas AP2 subunit overexpression induced Smad3 phosphorylation even in the absence of FLIL33. RNA-Seq transcriptomic analyses revealed that fibroblast stimulation with TGF-β induced major changes in expression levels of numerous genes, whereas overexpression of FLIL33 induced modest expression changes in a small number of genes. Furthermore, qRT-PCR tests demonstrated that despite inducing Smad3 phosphorylation, FLIL33 did not induce collagen gene transcription and even mildly attenuated TGF-β-induced levels of collagen I and III mRNAs. We conclude that FLIL33 induces Smad3 phosphorylation through a TGF-β-independent but TGF-β receptor- and AP2- dependent mechanism and has limited downstream transcriptomic consequences.

IL-33 已成为免疫、炎症和纤维化反应的中心介质。许多研究都集中在成熟的 IL-33 上，但前体全长 IL-33 (FLIL33) 的表达升高也与一系列疾病有关，包括组织纤维化。我们之前报道过，现在证实 FLIL33 的过表达诱导了来自健康供体和特发性肺纤维化患者的原代人肺成纤维细胞中 TGF-β 的关键促纤维化信号介质 Smad3 的磷酸化。目前，我们证明 FLIL33 诱导的 Smad3 磷酸化未被抗 TGF-β 抗体消除，但被 ALK5/TGFBR1 特异性和 Smad3 特异性抑制消除，表明 FLIL33 作用不依赖于 TGF-β，但依赖于其受体，TGFBR。蛋白质印迹分析显示，FLIL33 过表达增加了接头蛋白复合物 2（AP2）（一种已知的 TGFBR 结合伴侣）的 AP2A1 和 AP2B1 亚基的水平，但不影响亚细胞分布。siRNA 介导的对这些亚基的抑制阻断了 FLIL33 诱导的 Smad3 磷酸化，而 AP2 亚基过表达即使在没有 FLIL33 的情况下也能诱导 Smad3 磷酸化。RNA-Seq 转录组学分析显示，用 TGF-β 刺激成纤维细胞会引起许多基因表达水平的重大变化，而 FLIL33 的过表达会引起少数基因的适度表达变化。此外，qRT-PCR 测试表明，尽管诱导了 Smad3 磷酸化，FLIL33 不诱导胶原基因转录，甚至轻度减弱 TGF-β 诱导的胶原 I 和 III mRNA 水平。我们得出结论，FLIL33 通过 TGF-β 非依赖性但 TGF-β 受体和 AP2 依赖性机制诱导 Smad3 磷酸化，并且具有有限的下游转录组结果。