Chen Wei, Zhao Yue, Li Xian C, Kubiak Jacek Z, Ghobrial Rafik M, Kloc Malgorzata
The Houston Methodist Research Institute, Houston, TX, USA; Department of Nephrology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
The Houston Methodist Research Institute, Houston, TX, USA.
Int J Biochem Cell Biol. 2017 Dec;93:12-24. doi: 10.1016/j.biocel.2017.10.009. Epub 2017 Oct 20.
Macrophages play crucial role in tissue homeostasis and the innate and adaptive immune response. Depending on the state of activation macrophages acquire distinct phenotypes that depend on actin, which is regulated by small GTPase RhoA. The naive M0 macrophages are slightly elongated, pro-inflammatory M1 are round and M2 anti-inflammatory macrophages are elongated. We showed previously that interference with RhoA pathway (RhoA deletion or RhoA/ROCK kinase inhibition) disrupted actin, produced extremely elongated (hummingbird) macrophage phenotype and inhibited macrophage movement toward transplanted hearts. The RhoA function depends on the family of guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP and activate RhoA that reorganizes actin cytoskeleton. Using actin staining, immunostaining, Western blotting, flow cytometry and transmission electron microscopy we studied how a direct inhibition of Rho-GEFs with Rhosin (Rho GEF-binding domain blocker) and Y16 (Rho GEF DH-PH domain blocker) affects M0, M1 and M2 macrophage phenotypes. We also studied how Rho-GEFs inhibition and RhoA deletion affects organization of Golgi complex that is crucial for normal macrophage functions such as phagocytosis, antigen presentation and receptor recycling. We found that GEFs inhibition differently affected M0, M1 and M2 macrophages phenotype and that GEFs inhibition and RhoA deletion both caused changes in the ultrastructure of the Golgi complex. These results suggest that actin/RhoA- dependent shaping of macrophage phenotype has different requirements for activity of RhoA/GEFs pathway in M0, M1 and M2 macrophages, and that RhoA and Rho-GEFs functions are necessary for the maintenance of actin-dependent organization of Golgi complex.
巨噬细胞在组织稳态以及先天性和适应性免疫反应中发挥着关键作用。根据激活状态的不同，巨噬细胞会呈现出不同的表型，这些表型取决于由小GTP酶RhoA调节的肌动蛋白。未激活的M0巨噬细胞略显细长，促炎性M1巨噬细胞呈圆形，而抗炎性M2巨噬细胞则是细长的。我们之前的研究表明，干扰RhoA信号通路（RhoA缺失或RhoA/ROCK激酶抑制）会破坏肌动蛋白，产生极度细长（蜂鸟状）的巨噬细胞表型，并抑制巨噬细胞向移植心脏的移动。RhoA的功能依赖于鸟嘌呤核苷酸交换因子（GEF）家族，该家族催化GDP与GTP的交换并激活RhoA，进而重组肌动蛋白细胞骨架。我们使用肌动蛋白染色、免疫染色、蛋白质免疫印迹、流式细胞术和透射电子显微镜，研究了用Rhosin（Rho GEF结合域阻滞剂）和Y16（Rho GEF DH-PH域阻滞剂）直接抑制Rho-GEFs如何影响M0、M1和M2巨噬细胞表型。我们还研究了Rho-GEFs抑制和RhoA缺失如何影响高尔基体复合体的组织，而高尔基体复合体对于正常巨噬细胞功能（如吞噬作用、抗原呈递和受体循环利用）至关重要。我们发现，GEFs抑制对M0、M1和M2巨噬细胞表型的影响各不相同，并且GEFs抑制和RhoA缺失均导致高尔基体复合体超微结构的改变。这些结果表明，肌动蛋白/RhoA依赖性的巨噬细胞表型塑造对M0、M1和M2巨噬细胞中RhoA/GEFs信号通路的活性有不同要求，并且RhoA和Rho-GEFs的功能对于维持肌动蛋白依赖性的高尔基体复合体组织是必需的。
