Expression Analysis of Arpin, a Novel Arp2/3 Inhibitor, in Endothelial and Epithelial Cells under Basal and Inflammatory Conditions
Authors: Paredes Sandra Denisse Chanez , Cordero Julio Garcia , Robles Hilda Vargas , Barron Leticia Cedillo , Schnoor Michael 
Published: November 2023

The actin cytoskeleton regulates a wide range of cellular processes including cell migration, vesicular trafficking, phagocytosis, cytokinesis and barrier homeostasis. An important modulator of actin remodeling is the actin‐related protein 2/3 (Arp2/3) complex. Arp2/3 is a weak actin nucleator in vitro but it can be activated by direct interaction with different nucleation promoting factors. Other proteins can inhibit Arp2/3 including glia maturation factors (GMFs), coronins, cofilin, gadkin and protein interacting with C kinase 1 (PICK1). Recently a new Arp2/3 inhibitor has been identified and termed arpin that co‐localizes with Arp2/3 in mouse fibroblasts where it inhibits lamellipodial tip formation and cell migration. However, virtually nothing is known about expression and localization patterns of arpin in other cells or organs. Thus, we investigated arpin expression, localization and possible functions in barrier regulation using different endothelial and epithelial cells and mouse tissues. Arpin has two isoforms in humans and only one in mice. We found that both human arpin mRNA transcripts were expressed in similar amounts in endothelial HMEC‐1 and epithelial Caco‐2 cells, whereas in hematopoietic cells arpin expression corresponds to the shorter transcript 2 suggesting tissue‐specific expression of arpin transcripts. In mouse tissues the only known arpin isoform is expressed in kidney, heart, liver, colon and lung in comparable amounts. Since Arp2/3‐mediated actin remodeling occurs in endothelial and epithelial cells during inflammation, we analyzed effects of inflammation on the expression of arpin. We found that the mRNA levels of both variants decreased in HMEC‐1 and Caco‐2 cells after pro‐inflammatory cytokine treatment. In order to analyze arpin protein expression, we generated polyclonal and monoclonal antibodies. Immunofluorescence staining showed that arpin co‐localized with the Arp2/3 complex in the cytosol and at the cell periphery of Caco‐2 cells. Arpin also partly co‐localized with β‐catenin at adherens junctions and ZO‐1 at tight junctions, suggesting a possible role of arpin in the regulation of interepithelial junctions. A similar pattern was observed ex vivo in mouse lung cryosections where arpin also co‐localized with E‐cadherin. Importantly, under septic conditions, arpin expression strongly decreased and co‐localization with E‐cadherin was lost. In mouse colon tissue, arpin was located in the apical and basolateral regions of crypt intestinal epithelial cells, where it also co‐localized with Arp2/3 and ZO‐1. Interestingly, after induction of colitis by dextran sulfate sodium, we observed a relocalization of arpin including loss of co‐localization between arpin, Arp2/3 and ZO‐1. These data demonstrate that arpin is expressed in endothelial and epithelial cells and downregulated during inflammation. The localization of arpin at intercellular junctions in both endothelial and epithelial cells in vitro and ex vivo implies a role for arpin in the regulation of tissue barrier integrity.

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