Actin polymerization inhibition by targeting ARPC2 affects intestinal stem cell homeostasis.

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.