Crbn modulates calcium influx by regulating Orai1 during efferocytosis

Hyunji Moon,Gwangrog Lee,Byeongjin Moon,Seung-Joo Yang,Juyeon Lee,Daeho Park,Chanhyuk Min,Steve K Cho,Kwanhyeong Kim,Chang Sup Lee,Chul-Seung Park,Sang-Ah Lee,Deokhwan Kim,Gayoung Kim,Susumin Yang

doi:10.1038/s41467-020-19272-0

Abstract

Calcium flux regulating intracellular calcium levels is essential and modulated for efficient efferocytosis. However, the molecular mechanism by which calcium flux is modulated during efferocytosis remains elusive. Here, we report that Orai1, a Crbn substrate, is upregulated via its attenuated interaction with Crbn during efferocytosis, which increases calcium influx into phagocytes and thereby promotes efferocytosis. We found that Crbn deficiency promoted phagocytosis of apoptotic cells, which resulted from facilitated phagocytic cup closure and was nullified by a CRAC channel inhibitor. In addition, Orai1 associated with Crbn, resulting in ubiquitination and proteasomal degradation of Orai1 and alteration of SOCE-mediated calcium influx. The association of Orai1 with Crbn was attenuated during efferocytosis, leading to reduced ubiquitination of Orai1 and consequently upregulation of Orai1 and calcium influx. Collectively, our study reveals a regulatory mechanism by which calcium influx is modulated by a Crbn-Orai1 axis to facilitate efferocytosis.

Highlights

Calcium flux regulating intracellular calcium levels is essential and modulated for efficient efferocytosis
Previous studies showing that Ampk is activated during efferocytosis and in Crbn-depleted cells led us to investigate whether Crbn affects engulfment of apoptotic cells[27,33,34]
The number of apoptotic cells ingested per phagocyte was lower for Crbn-overexpressing cells than for control cells, as indicated by the mean fluorescence intensity (MFI, which represented the relative number of apoptotic cells per phagocyte) (Fig. 1a and Supplementary Fig. 1a, b)

Summary

Introduction

Calcium flux regulating intracellular calcium levels is essential and modulated for efficient efferocytosis. Orai[1] associated with Crbn, resulting in ubiquitination and proteasomal degradation of Orai[1] and alteration of SOCE-mediated calcium influx. This binding alters the substrate specificity of Crbn, resulting in degradation of neo-substrates, such as IKZF1, CK1a, and GSPT1 or decreased degradation of its endogenous substrates such as MEIS223,24,30–32 These alterations are considered to underlie the immunomodulatory effects and teratogenic activity of IMiDs. The activation of Ampk in Crbn-depleted cells and during efferocytosis led us to question whether Crbn influences the clearance of apoptotic cells[26,27,33,34,35]. The interaction of Orai[1] with Crbn was weakened during efferocytosis, which reduced ubiquitination and increased the level of Orai[1] These effects increased calcium influx and the efficiency of efferocytosis. This study demonstrates unappreciated machinery that modulates calcium influx to promote removal of apoptotic cells through a Crbn–Orai[1] axis in efferocytosis

Methods

Results

Conclusion