Department of Physiology, Pathophysiology & Toxicology and ZBAF (Centre for Biomedical Education and Research), Faculty of Health, School of Medicine, Witten/Herdecke University, Stockumer Str 12 (Thyssenhaus), D-58453, Witten, Germany.
Department of Medicine, Hematology and Oncology, Martin Luther University Halle-Wittenberg, 06120, Halle (Saale), Germany.
Cell Commun Signal. 2018 Nov 7;16(1):74. doi: 10.1186/s12964-018-0285-3.
We have previously evidenced apical expression of the 24p3/NGAL/lipocalin-2 receptor (Lcn2-R; SLC22A17) in inner medullary collecting duct (IMCD) cells, which are present in vivo in a hyperosmotic/-tonic environment that activates canonical Wnt/β-catenin signaling. The localization of Lcn2-R in the inner medulla is intriguing considering local bacterial infections trigger toll-like receptor-4 (TLR-4)-mediated secretion of the bacteriostatic Fe-free (apo-)Lcn2.
To determine the effects of osmolarity/tonicity changes, Wnt/β-catenin and TLR-4 activation on Lcn2-R and Lcn2 expression and cell viability in rat primary IMCD and mouse (m)IMCD3 cells.
Normosmolarity/-tonicity was 300 mosmol/l whereas hyperosmolarity/-tonicity was induced by adding 100 mmol/l NaCl + 100 mmol/l urea (600 mosmol/l, 1-7 days). Lcn2-R and Lcn2 expression were determined by qPCR, immunoblotting, flow cytometry and immunofluorescence microscopy. β-catenin was silenced by RNAi. Cell viability/death was determined with MTT and LDH release assays. TLR-4 was activated by bacterial lipopolysaccharides (LPS).
Hyperosmotic/-tonic media upregulated Lcn2-R by ~4-fold and decreased Lcn2 expression/secretion, along with Wnt/β-catenin activation, in IMCD cells. These effects of hyperosmotic/-tonic media on Lcn2-R/Lcn2 expression were reverted by normosmolarity/-tonicity, β-catenin silencing and/or LPS. Exposure of cells with endogenous or stably overexpressing Lcn2-R to apo-Lcn2 or LPS decreased cell viability.
Lcn2-R upregulation and Lcn2 downregulation via Wnt/β-catenin may promote adaptive osmotolerant survival of IMCD cells in response to hyperosmolarity/-tonicity whereas Lcn2 upregulation and Lcn2-R downregulation via TLR-4 and/or normosmolarity/-tonicity may protect IMCD cells against bacterial infections and prevent autocrine death induction by Lcn2.
我们之前已经证明，24p3/NGAL/脂联素-2 受体（Lcn2-R；SLC22A17）在内髓集合管（IMCD）细胞中的顶端表达，这些细胞在体内存在于激活经典 Wnt/β-连环蛋白信号的高渗/高渗环境中。考虑到局部细菌感染会触发 Toll 样受体 4（TLR-4）介导的抑菌无铁（apo-）Lcn2 的分泌，Lcn2-R 在髓质中的定位很有趣。
确定渗透压/张力变化、Wnt/β-连环蛋白和 TLR-4 激活对大鼠原代 IMCD 和小鼠（m）IMCD3 细胞中 Lcn2-R 和 Lcn2 表达和细胞活力的影响。
正常渗透压/张力为 300 mosmol/l，而高渗透压/张力通过添加 100 mmol/l NaCl + 100 mmol/l 尿素（600 mosmol/l，1-7 天）诱导。通过 qPCR、免疫印迹、流式细胞术和免疫荧光显微镜检测 Lcn2-R 和 Lcn2 的表达。β-连环蛋白通过 RNAi 沉默。用 MTT 和 LDH 释放试验测定细胞活力/死亡。通过细菌脂多糖（LPS）激活 TLR-4。
高渗/高渗培养基使 IMCD 细胞中 Lcn2-R 上调约 4 倍，并降低 Lcn2 的表达/分泌，同时激活 Wnt/β-连环蛋白。高渗/高渗培养基对 Lcn2-R/Lcn2 表达的这些影响可通过正常渗透压/张力、β-连环蛋白沉默和/或 LPS 逆转。用内源性或稳定过表达 Lcn2-R 的细胞暴露于 apo-Lcn2 或 LPS 会降低细胞活力。
Wnt/β-连环蛋白介导的 Lcn2-R 上调和 Lcn2 下调可能促进 IMCD 细胞对高渗/高渗的适应性耐受力，而 TLR-4 和/或正常渗透压/张力介导的 Lcn2 上调和 Lcn2-R 下调可能保护 IMCD 细胞免受细菌感染，并防止 Lcn2 诱导的自分泌死亡。
