CRELD2 is a novel modulator of calcium mobilisation and calcineurin-NFAT signalling during osteoclast differentiation

Cysteine rich with epidermal growth factor (EGF)-like domains 2 (CRELD2) is an endoplasmic reticulum (ER) resident chaperone protein with calcium binding properties. CRELD2 is an ER-stress regulated gene that has been implicated in the pathogenesis of skeletal dysplasias and has been shown to play an important role in the differentiation of chondrocytes and osteoblasts. Despite CRELD2 having an established role in skeletal development and bone formation, its role in osteoclasts is currently unknown. Here we show for the first time that CRELD2 plays a novel role in trafficking transforming growth factor beta 1 (TGF-ß1), which is linked to an upregulation in the expression of Nfat2, the master regulator of osteoclast differentiation in early osteoclastogenesis. Despite this finding, we show that CRELD2 is dispensable for osteoclastogenesis. Indeed, the overexpression of Creld2 impaired osteoclast differentiation due to a reduction in the activity of the calcium-dependent phosphatase calcineurin. This in turn led to a subsequent block in the dephosphorylation of nuclear factor of activated T cells 1 (NFATc1) dephosphorylation preventing its nuclear localisation and activation as a pro-osteoclastogenic transcription factor. Our exciting results show that the overexpression of Creld2 in osteoclasts impaired calcium mobilisation which is essential for activating calcineurin and promoting osteoclastogenesis. Therefore, our data proposes a novel inhibitory role for this calcium-binding ER-resident chaperone in modulating calcium flux during osteoclast differentiation which has important implications in our understanding of bone remodelling and the pathogenesis of skeletal diseases. Overall design: RNA was extracted from CONTROL and CRELD2 overexpressing RAW264.7 osteoclasts 48 hours after RANKL stimulation using Promega ReliaPrep Cell Miniprep System following the manufacturer's protocol, and was sequenced using an Illumina NextSeq 500.