A recent study has shown that CRELD2 plays an important role in skeletal development and bone homeostasis by promoting the differentiation of chondrocytes and osteoblasts19. Despite the well-established role of CRELD2 in bone-forming osteoblasts, its role in osteoclast differentiation is unknown. Since CRELD2 is expressed in the skeleton and plays a role in bone homeostasis, understanding its role in osteoclasts would provide further understanding of the complex process of bone remodelling, which would have an impact on our understanding of associated diseases.
Here, we show that CRELD2 is dispensable for osteoclastogenesis and its expression is decreased upon stimulation of osteoclast differentiation. Indeed, we show that overexpressing CRELD2 in a pre-osteoclast cell line impairs osteoclast differentiation and activity as determined by a significant reduction in osteoclast size, number of nuclei and resorptive activity. This was confirmed by transcriptomic analyses that showed a downregulation in the expression of multiple osteoclastogenic marker genes in CRELD2 OE osteoclasts.
Although overexpressing CRELD2 in pre-osteoclasts inhibited osteoclast differentiation, we were surprised to find that the expression of Nfat2, encoding NFATc1 the master regulator of osteoclastogenesis, was significantly upregulated in CRELD2 OE osteoclasts 24 hours after RANKL stimulation. To understand how CRELD2 overexpression resulted in increased Nfat2 expression, we referred to results from a previous study that identified putative CRELD2 binding partners in two independent cell types by co-immunoprecipitation. Of note, CRELD2 was found to bind to TGF-β1. Previous studies have shown that TGF-β1 plays a complex and controversial role in osteoclastogenesis depending on the model system. For example, in co-culture with osteoblasts TGF-β downregulates RANKL expression and inhibits osteoclastogenesis, whereas it has been shown to directly stimulate commitment and osteoclastogenic differentiation of RAW264.7 cells by upregulating RANK expression5,26,27. In addition, a study has shown that TGF-β directly upregulates Nfat2 expression within 24 hours of RANKL stimulation24. Here, TGF-β was shown to commit monocytes to the osteoclast lineage in early differentiation; however, it performs an inhibitory role in the latter stages of osteoclastogenesis24.
Since we confirmed CRELD2 bound to TGF-β1 in RAW264.7 pre-osteoclasts and previous cellular functions of CRELD2 demonstrated a role in protein folding and trafficking, we hypothesised that CRELD2 promotes the secretion of TGF-β1 from RAW264.7 cells. Indeed, we show here for the first time that CRELD2 overexpression promoted the extracellular trafficking of TGF-β1 resulting in increased TGF-β signalling in CRELD2 OE osteoclasts. We therefore hypothesised that the upregulation of Nfat2 expression in CRELD2 OE osteoclasts 24 hours after RANKL stimulation was attributed to increased TGF-β secretion. Despite the initial increase in Nfat2 expression, by 48 hours post-RANKL treatment the levels of Nfat2 were comparable between control and CRELD2 OE osteoclasts, further indicating that TGF-β1 functions to prime pre-osteoclasts during the early stages of differentiation.
Inactive NFATc1 exists in the cytoplasm in a phosphorylated form. To be active as a transcription factor it undergoes dephosphorylation by the calcium-dependant phosphatase calcineurin and is translocated to the nucleus where it regulates the expression of genes that drive osteoclastogenesis. To understand how osteoclastogenesis was impaired following CRELD2 overexpression, despite the upregulation of Nfat2 in CRELD2 OE osteoclasts in early differentiation, we analysed NFATc1 activation and nuclear translocation. It is important to note that although the expression of Nfat2 was upregulated in CRELD2 OE osteoclasts 24 hours after RANKL stimulation, this was not reflected at the protein level. In fact, total NFATc1 levels were reduced following CRELD2 overexpression, which resulted in an increase in the ratio of phosphorylated NFATc1 to total NFATc1, indicating that the transcriptional activity of NFATc1 in CRELD2 OE was decreased. This can be attributed to a reduction in the activity of calcineurin following CRELD2 overexpression, which in turn resulted in a reduction in nuclear localisation of NFATc1.
We therefore sought to understand how overexpression of this ER-stress inducible and ER-resident calcium-binding protein affected the activity of cytoplasmic calcineurin. Studies have shown that other calcium-binding ER-resident proteins regulate calcium mobilisation, which is required for the activation of calcineurin. Indeed, the ER-stress regulated calcium binding chaperones, calreticulin and calnexin, have been shown to modulate and reduce cytoplasmic calcium flux8–10. Indeed, calreticulin expression has been linked to reduced osteoclast differentiation10 due to impaired calcium flux. We therefore sought to investigate the effects of CRELD2 expression on calcium mobilisation during osteoclastogenesis. Similar to findings from other studies, our data showed that the overexpression of the calcium-binding ER-resident chaperone protein CRELD2 resulted in a reduction in the overall level of calcium released from the ER. Recent work has shown that CRELD2 interacts with the ER-resident calcium binding protein reticulocalbin (RCAN) 1, which mediates calcium-dependent cellular activities and inhibits calcium release from the ER through binding to the calcium channel inositol 1,4,5-trisphosphate (IP3) receptor type1 (IP3R1). Based on these recent findings, we hypothesise that CRELD2 mediates calcium flux via interactions with RCAN1; however, the relationship between these two calcium-binding ER proteins requires further study.
In summary, our results propose a novel inhibitory role of CRELD2 during osteoclastogenesis. We show for the first time that although CRELD2 promotes the trafficking of TGF-β1, osteoclastogenesis is disrupted by CRELD2 expression. We also demonstrated that CRELD2 expression blocks calcium release from the ER, impairing osteoclast differentiation due to reduced calcium-dependant calcineurin activity and the subsequent nuclear translocation of NFATc1, the master regulator of osteoclastogenesis.
Although no mutations have been identified in CRELD2 to date, these data highlight a novel role for CRELD2 in osteoclastogenesis and indicates CRELD2 may be a potential genetic locus for skeletal diseases caused by dysregulated osteoclast differentiation and function.