Sorting protein VPS33B regulates exosomal autocrine signaling to mediate hematopoiesis and leukemogenesis
Hao Gu, … , Guo-Qiang Chen, Junke Zheng
Hao Gu, … , Guo-Qiang Chen, Junke Zheng
Published October 31, 2016
Citation Information: J Clin Invest. 2016;126(12):4537-4553. https://doi.org/10.1172/JCI87105.
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Research Article Hematology Stem cells

Sorting protein VPS33B regulates exosomal autocrine signaling to mediate hematopoiesis and leukemogenesis

Abstract

Certain secretory proteins are known to be critical for maintaining the stemness of stem cells through autocrine signaling. However, the processes underlying the biogenesis, maturation, and secretion of these proteins remain largely unknown. Here we demonstrate that many secretory proteins produced by hematopoietic stem cells (HSCs) undergo exosomal maturation and release that is controlled by vacuolar protein sorting protein 33b (VPS33B). Deletion of VPS33B in either mouse or human HSCs resulted in impaired exosome maturation and secretion as well as loss of stemness. Additionally, VPS33B deficiency led to a dramatic delay in leukemogenesis. Exosomes purified from either conditioned medium or human plasma could partially rescue the defects of HSCs and leukemia-initiating cells (LICs). VPS33B co-existed in exosomes with GDI2, VPS16B, FLOT1, and other known exosome markers. Mechanistically, VPS33B interacted with the GDI2/RAB11A/RAB27A pathway to regulate the trafficking of secretory proteins as exosomes. These findings reveal an essential role for VPS33B in exosome pathways in HSCs and LICs. Moreover, they shed light on the understanding of vesicle trafficking in other stem cells and on the development of improved strategies for cancer treatment.

Authors

Hao Gu, Chiqi Chen, Xiaoxin Hao, Conghui Wang, Xiaocui Zhang, Zhen Li, Hongfang Shao, Hongxiang Zeng, Zhuo Yu, Li Xie, Fangzhen Xia, Feifei Zhang, Xiaoye Liu, Yaping Zhang, Haishan Jiang, Jun Zhu, Jiangbo Wan, Chun Wang, Wei Weng, Jingjing Xie, Minfang Tao, Cheng Cheng Zhang, Junling Liu, Guo-Qiang Chen, Junke Zheng

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Figure 1

Stemness-related secretory proteins exist in exosomes.

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Stemness-related secretory proteins exist in exosomes. (A and B) 293T ce...
(A and B) 293T cells transfected with TPO, ANGPTL2, and ANGPTL3 were costained with either α-tubulin (A) or CD63 (B) using immunofluorescence staining to evaluate the colocalization, as shown in the merged images of each row. Nuclei were counterstained with DAPI. Scale bars: 10 μm. (C) Exosomes were purified from TPO-, ANGPTL2- and ANGPTL3-conditioned medium (CM) of 293T cells and immunoblotted with antibodies against V5 (for TPO), Flag (for ANGPTL2 and ANGPTL3), and TSG101 and FLOT1 (exosome markers). Ctrl, empty vector; BE, before extraction; AE, after extraction. (D) Representative transmission electron micrographs of exosomes purified from ANGPTL2-conditioned medium of 293T cells (scale bars: 100 nm). (E) Quantitative data for the levels of TPO-, ANGPTL2-, and ANGPTL3-containing exosomes in supernatant after differential centrifugation (AE/BE, n = 3). (F) Exosomes were purified from human plasma and assayed to detect TPO, ANGPTL2, ANGPTL3, TSG101, FLOT1, VPS16B, and VPS33B. (G) Quantitative data for the levels of TPO-, ANGPTL2-, and ANGPTL3-containing exosomes in human plasma after differential centrifugation (AE/BE, n = 3). Experiments were conducted 3 to 5 times for validation.