Orai1‐mediated calcium entry plays a critical role in osteoclast differentiation and function by regulating activation of the transcription factor NFATc1

Abstract

Bone diseases such as postmenopausal osteoporosis are primarily caused by excessive formation and activity of osteoclasts (OCLs). Receptor activator of nuclear factor-κB ligand (RANKL) is a key initiating cytokine for OCL differentiation and function. RANKL induces calcium (Ca(2+)) oscillations, resulting in selective and robust induction of nuclear factor of activated T cells c1 (NFATc1), a Ca(2+)-responsive transcription factor that drives osteoclastogenesis. Store-operated Ca(2+) entry (SOCE) is a major Ca(2+) influx pathway in most nonexcitable cell types and is activated by any stimulus that depletes Ca(2+) stores in the endoplasmic reticulum. Although the role of Orai1, a SOCE channel in the plasma membrane, in maintaining Ca(2+) oscillations and transactivation of NFAT in other cell types is well known, its contribution to osteoclastogenesis remains unclear. We show here that silencing of the Orai1 gene with viral delivery of shRNA reduces SOCE and inhibits RANKL-induced osteoclastogenesis of RAW264.7 cells, a murine monocyte/macrophage cell line, by suppressing the induction of NFATc1. This was accompanied by defective induction of OCL-specific genes, such as tartrate-resistant acid phosphatase and immunoreceptor OCL-associated receptor, which are known to be direct transcriptional targets of NFATc1 during osteoclastogenesis. In addition, maturation of OCLs was abrogated by defective cell fusion of pre-OCLs depleted of Orai1, consistent with defective RANKL-mediated induction of d2 isoform of vacuolar ATPase V(o) domain that is involved in cell fusion of pre-OCLs. We found that the functional bone resorbing capacity was severely impaired in OCLs depleted of Orai1, potentially related to the observed decrease in the induction of cathepsin K, a major bone matrix degrading protease. Our results indicate that Orai1 plays a critical role in the differentiation and function of OCLs, suggesting that Orai1 might be a potential therapeutic target for the treatment or prevention of bone loss caused by OCLs.

A TRPC1 Protein-dependent Pathway Regulates Osteoclast Formation and Function

“…In regard to the genetic interaction of Trpc1 and I-mfa in osteoclastogenesis, we propose that maximal and/or persistent activation of I CRAC /I SOC through TRPC1 in cells lacking I-mfa leads to excessive osteoclastogenesis and reduced bone mass. This suggestion is supported by our experiments in myeloid precursors and in studies in Orai1 null mice showing similar but more severely defective osteoclastogenesis (51) and in vitro studies using Orai1-depleted osteoclasts (52,53). The more severe and nonspecific effect of the deletion of Orai1 in numerous cell types, including osteoclasts compared with TRPC1, is in agreement with Orai1 being a core component of the CRAC channel and TRPC1 being a regulatory protein whose function is dispensable for Orai1.…”

Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass

“…Next we considered the protein Stim1, which is an ER Ca 2+ sensor controlling Ca 2+ fluxes during osteoclastogenesis (16,17,25). We expressed Tmem178-HA and Stim1-Myc in HEK293T cells and found that Tmem178 and Stim1 interact in resting conditions (control), and to a less extent in the presence of Tg and Tg + 1.8 mM Ca 2+ (Fig.…”

“…Targeted deletion of PLCγ2 in mice results in osteopetrosis due to insufficient NFATc1 expression. Similarly, genetic or pharmacological interference of ER or plasma membrane Ca 2+ channel activity blocks osteoclastogenesis in vitro and in vivo by impairing NFATc1 expression (14)(15)(16)(17)(18)(19). Differently from T cells, however, NFATc1 activation must be sustained throughout osteoclastogenesis over the course of days and depends on both amplitude and duration of the Ca 2+ fluxes.…”

Role of STIM1- and Orai1-mediated Ca2+ entry in Ca2+-induced epidermal keratinocyte differentiation

“…As its name suggests, SOCE is activated by depletion of Ca 2+ stores in the endoplasmic reticulum (ER). SOCE is known to be involved in cell proliferation and differentiation processes (Darbellay et al, 2009;Hwang and Putney, 2012;Johnstone et al, 2010). SOCE is mediated essentially by two classes of proteins, the STIM and Orai proteins (Feske et al, 2006; (Liou et al, 2005).…”