Actin polymerization inhibition by targeting ARPC2 affects intestinal stem cell homeostasis

Ruzhen Zhang; Sheng Chen; Zhifan Yang; Ning Zhang; Kenan Guo; Keyi Lv; Zimo Zhou; Meijiao Gao; Xiancheng Hu; Yongping Su; Jianming He; Fengchao Wang

doi:10.1093/burnst/tkad038

Actin polymerization inhibition by targeting ARPC2 affects intestinal stem cell homeostasis

Burns Trauma. 2023 Oct 16:11:tkad038. doi: 10.1093/burnst/tkad038. eCollection 2023.

Authors

Ruzhen Zhang^{1

2}, Sheng Chen¹, Zhifan Yang¹, Ning Zhang¹, Kenan Guo^{1

3}, Keyi Lv⁴, Zimo Zhou¹, Meijiao Gao¹, Xiancheng Hu², Yongping Su¹, Jianming He⁵, Fengchao Wang¹

Affiliations

¹ State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China.
² College of Life Sciences, Chongqing Normal University，Chongqing, 401331China.
³ Department of Laboratory Animal Science, College of Basic Medical Sciences, Army Medical University, Third Military Medical University, Chongqing 400038, China.
⁴ Department of Military Cognitive Psychology, School of Psychology, Third Military Medical University, Chongqing, 400038, China.
⁵ Department of Radiotherapy, Hebei Province Hospital of Chinese Medicine, Hebei University of Chinese Medicine, Key Laboratory of Integrated Chinese and Western Medicine for Gastroenterology Research (Hebei), Shijiazhuang, 050011.

Abstract

Background: The rapid turnover of the intestinal epithelium is driven by the proliferation and differentiation of intestinal stem cells (ISCs). The dynamics of the F-actin cytoskeleton are critical for maintaining intercellular force and the signal transduction network. However, it remains unclear how direct interference with actin polymerization impacts ISC homeostasis. This study aims to reveal the regulatory effects of the F-actin cytoskeleton on the homeostasis of intestinal epithelium, as well as the potential risks of benproperine (BPP) as an anti-tumor drug.

Methods: Phalloidin fluorescence staining was utilized to test F-actin polymerization. Flow cytometry and IHC staining were employed to discriminate different types of intestinal epithelial cells. Cell proliferation was assessed through bromo-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays. The proliferation and differentiation of intestinal stem cells were replicated in vitro through organoid culture. Epithelial migration was evaluated through BrdU pulse labeling and chasing in mice.

Results: The F-actin content was observed to significantly increase as crypt cells migrated into the villus region. Additionally, actin polymerization in secretory cells, especially in Paneth cells (PCs), was much higher than that in neighboring ISCs. Treatment with the newly identified actin-related protein 2/3 complex subunit 2 (ARPC2) inhibitor BPP led to a dose-dependent increase or inhibition of intestinal organoid growth in vitro and crypt cell proliferation in vivo. Compared with the vehicle group, BPP treatment decreased the expression of Lgr5 ISC feature genes in vivo and in organoid culture. Meanwhile, PC differentiation derived from ISCs and progenitors was decreased by inhibition of F-actin polymerization. Mechanistically, BPP-induced actin polymerization inhibition may activate the Yes1-associated transcriptional regulator pathway, which affects ISC proliferation and differentiation. Accordingly, BPP treatment affected intestinal epithelial cell migration in a dose-dependent manner.

Conclusion: Our findings indicate that the regulation of cytoskeleton reorganization can affect ISC homeostasis. In addition, inhibiting ARPC2 with the Food and Drug Administration-approved drug BPP represents a novel approach to influencing the turnover of intestinal epithelial cells.

Keywords: ARPC2; Benproperine; F-actin; Intestinal stem cell; YAP.