Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

Abstract

Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcRγ) and DNAX-activating protein 12 kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin (β3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10 days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that FK506 treatment significantly (p < 0.05) reduced the expression of NFATc1, CathK, OSCAR, FcRγ, TREM2 and DAP12 during the terminal stage of osteoclast formation. VIVIT treatment significantly (p < 0.05) decreased CathK, OSCAR, FcRγ, and AnnVIII, gene expression. This data suggest FK506 and VIVIT act differently in targeting the calcineurin-NFAT signalling cascade to suppress key mediators of the ITAM pathway during late stage osteoclast differentiation and this is associated with a reduction in both osteoclast differentiation and activity.

Introduction

High numbers of bone resorbing osteoclasts cells have been identified at the sites of bone loss in a number of diseases, such as near arthritic bone erosions in RA [1] and peri-implant osteolysis [2]. Osteoclast differentiation requires receptor activator of NF-κβ ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). RANKL expression by bone stromal cells such as osteoblasts, synovial fibroblasts and T cell is increased adjacent to sites of pathological bone loss [3], [4], [5]. RANKL binds to its receptor RANK, expressed on pre-osteoclasts to induce osteoclast differentiation and activation via elevated expression of the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) [6]. Activation of NFATc1 is mediated by the calcium ion dependent protein phosphatase, calcineurin, which dephosphorylates NFATc1 enabling its translocation to the nucleus [7]. Absence of NFATc1 retards osteoclastogenesis [8] and over-expression [9] or ectopic expression of constitutively active NFATc1 [6] stimulates osteoclastogenesis in the absence of RANKL. NFATc1 directly induces osteoclast-specific genes, including cathepsin K (CathK), tartrate resistant acid phosphatase (TRAP), calcitonin receptor (CTR), osteoclast-associated receptor (OSCAR) and β3 integrin [8], [10], [11], [12], [13], [14], [15], as well as auto-amplifies its own expression [9]. Additionally, the late stage substrate regulated osteoclast factor Annexin VIII (AnnVIII) [16] is also likely to be regulated by NFATc1 (unpublished data). Taken together, the calcineurin-NFATc1 pathway is pivotal to osteoclastogenesis.

Recently other co-stimulatory pathways involving NFATc1 have been identified in osteoclasts. The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway stimulates calcium signals that activate the calcineurin pathway and thus induce NFATc1 [14]. ITAM-containing adaptor proteins, DNAX-activating protein of molecular mass 12 kDa (DAP12) and Fc receptor common gamma chain (FcRγ) bind with their co-receptors; triggering receptor expressed in myeloid cells (TREM2) and osteoclast associated receptor (OSCAR) respectively to induce ITAM signalling [17], [18]. We have demonstrated increased expression of these factors adjacent to sites of bone loss in peri-implant loosening [19].

The importance of the ITAM pathway is particularly evident in Nasu-Hakola disease in humans, where a mutation of TREM2 or DAP12 leads to the development of bone cysts [20]. Importantly, mice lacking DAP12 or FcRγ have suppressed NFATc1 expression resulting and defective osteoclast formation [21]. Additionally, DAP12^−/− FcRγ^−/− mice develop osteopetrosis [22]. Osteoclast differentiation is impeded and bone resorption is remarkably reduced by mutations in DAP12 and TREM2 that result in their loss of function [20]. These studies demonstrate that the ITAM molecules, FcRγ, TREM2 and DAP12 play an important role in osteoclast development and activity.

OSCAR is induced via NFATc1 and OSCAR signalling via the FcRγ adaptor molecule further enhances ITAM signalling on ligand interaction [10], [14]. Genetic variations in OSCAR identify it as a potential underlying factor in postmenopausal osteoporosis [23]. Further to this, levels of cell bound and soluble OSCAR are modulated in diseases such as RA [24], [25].

The convergence of RANKL-RANK and the ITAM pathways at calcineurin/NFATc1 signalling identifies this interaction as a key therapeutic target for the treatment of osteoclast mediated bone loss [21]. Tacrolimus (FK506) has been identified as an effective treatment of DMARD-resistant or intolerant RA patients [26]. FK506 inhibits the phosphatase activity of calcineurin thus inhibiting translocation and the nuclear localisation of NFATc1 [6]. It has been shown to prevent RANKL-induced osteoclastogenesis in vitro [6], [27], [28] as well as having anabolic effects on the osteoblast in vivo and in vitro [29].

Side effects associated with treatment with FK506 led to the development of 11R-VIVIT peptide (MAGPHPVIVITGPHEE) (VIVIT) [30]. VIVIT is a more selective and potent inhibitor of the calcineurin-NFAT pathway as VIVIT interferes selectively with calcineurin-NFAT interaction without affecting calcineurin phosphatase activity [31]. We have reported that VIVIT affects osteoclast morphology and suppression of β3 integrin gene expression in murine bone marrow derived osteoclasts [13]. VIVIT has been shown to inhibit titanium particle induced bone resorption and expression of NFATc1 in bone marrow macrophages (BMMs)-derived osteoclasts [28], [32].

ITAM-related molecules appear to play a pivotal role in promoting osteoclast differentiation by co-stimulating the calcineurin-NFAT pathway. Therefore, we used FK506 and VIVIT to inhibit calcineurin-NFATc1 signalling assessed their effects on expression of ITAM associated molecules, osteoclastogenic genes and bone resorptive activity during osteoclast differentiation in vitro.