MFG-E8, a novel homeostatic regulator of osteoclastogenesis

Abstract

Although the glycoprotein MFG-E8 (milk fat globule-epidermal growth factor-factor 8) has been investigated extensively as an anti-inflammatory and homeostatic molecule, a possible role in bone homeostasis and disease was not addressed until recently. Our group has now shown that MFG-E8 is expressed by human and mouse osteoclasts and regulates their differentiation and function (Abe et al., J Immunol 2014;193:1383–1391). Whereas genetic deficiency or antibody-mediated neutralization of MFG-E8 enhances osteoclastogenesis and promotes inflammation-induced bone loss in mice, local administration of recombinant MFG-E8 blocks bone loss. These findings establish MFG-E8 as a novel homeostatic regulator of osteoclastogenesis and suggest that MFG-E8 could be exploited therapeutically to treat disorders associated with inflammatory bone loss, such as periodontitis and rheumatoid arthritis.

Introduction

The milk fat globule-epidermal growth factor (EGF)-factor 8 (MFG-E8; also known as lactadherin) is a secreted glycoprotein expressed in a range of tissues by a variety of cells including macrophages, fibroblasts, dendritic and epithelial cells ^[1]. In addition to a N-terminal signal peptide required for secretion, the molecule consists of two N-terminal EGF-like domains and two C-terminal discoidin-like domains with sequence homology to blood coagulation factors V and VIII ^[2]. MFG-E8 was shown to promote the phagocytosis of apoptotic cells by acting as an opsonin that bridges phosphatidylserine on apoptotic cells (bound by the C-terminal discoidin-like domains of MFG-E8) to αvβ3 integrin on phagocytes (bound by an RGD motif in the N-terminal region of MFG-E8) ^[3]. Efficient apoptotic cell clearance can prevent secondary necrosis and unwarranted inflammation. Moreover, MFG-E8 was shown to have direct anti-inflammatory properties and to suppress inflammatory tissue damage in several disease models ^[4]. In both humans and animal models, the expression of MFG-E8 declines considerably in inflammatory conditions ^[1], suggesting the potential use of MFG-E8 as a biomarker. In this regard, a recent study has demonstrated a negative association between the serum levels of MFG-E8 and the severity of coronary artery stenosis ^[5]. The authors suggested that MFG-E8 could serve as a marker of vascular complications or even be considered as a new therapeutic approach for atherosclerosis.

MFG-E8 is structurally similar with developmental endothelial locus-1 (Del-1) ^[2], an endothelial cell-secreted glycoprotein that regulates inflammatory cell recruitment ^[6,7]. MFG-E8 and Del-1 share about 50% amino-acid identity and Del-1 contains an additional EGF domain (i.e., it has three EGF and two discoidin-like domains). Our group has recently shown that Del-1 acts homeostatically to regulate local inflammation in periodontitis ^[8], a biofilm-induced inflammatory disease causing loss of bone support of the dentition ^[9]. Because of its documented anti-inflammatory effects and its structural similarity with Del-1, MFG-E8 attracted the attention of our research group and we thus set out to determine its role in periodontitis ^[10]. To this end, we used the ligature-induced periodontitis model in mice, where the placement of silk ligatures around molar teeth facilitates bacteria-mediated inflammation and bone loss ^[11].

Osteoclasts express and release MFG-E8

In the first experiment, we monitored the expression of MFG-E8 mRNA in the periodontal tissue. Consistent with the demonstrated downregulation of MFG-E8 expression in several models of inflammation ^[1], the periodontal MFG-E8 mRNA levels were drastically decreased within 24h following placement of the ligatures. Subsequently, and quite unexpectedly, the expression of MFG-E8 mRNA exhibited progressive elevation for several days (until day 8). The resurgence of MFG-E8 expression correlated with the appearance of osteoclasts (OCLs), giant multinucleated cells (MNCs) that resorb bone during physiological bone remodeling but also under pathologic inflammatory conditions (e.g., rheumatoid arthritis and periodontitis) that greatly potentiate their resorptive activity ^[12,13]. When the numbers of OCLs in the periodontal tissue dropped (from day 8 to day 10), so did the expression of MFG-E8. Moreover, we detected MFG-E8 protein in regions coinciding with the expression of cathepsin K (the predominant osteoclastic protease) and the presence of tartrate-resistant acid phosphatase (TRAP) positive MNCs.

The stunning spatial and temporal correlation of MFG-E8 re-expression with osteoclastogenesis prompted us to examine the intriguing possibility that MFG-E8 may derive from OCLs in the course of periodontitis. Indeed, we showed for the first time that OCLs express and release MFG-E8 protein, as shown by cell immunofluorescence, immunoblotting of whole-cell lysates, and immunoprecipitation from culture supernatants. MFG-E8 protein was detected in three different systems of receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis, including osteoclasts generated from RAW264.7 cells, mouse bone marrow-derived precursors, and human CD14+ monocytes.

MFG-E8 regulates OCL differentiation and function in vitro and in vivo

To characterize the role of MFG-E8 in osteoclastogenesis, we generated OCLs from wild-type (WT) or MFG-E8-deficient (Mfge8^−/−) osteoclast precursors (OCPs) from the bone marrow of mice. Mfge8^−/− OCPs underwent more efficient osteoclastogenesis than WT OCPs, consistent with higher expression of OCL differentiation and functional markers. Moreover, Mfge8^−/− OCLs caused enhanced resorption pit formation in comparison to their WT counterparts. Furthermore, exogenously added recombinant MFG-E8 inhibited RANKL-induced expression of OCL markers (NFATc1, β3 integrin, and cathepsin K), osteoclastogenesis from mouse or human osteoclast precursors, and resorption pit formation. These data indicate that MFG-E8 is a novel negative regulator of osteoclastogenesis, at least in vitro.

To test the relevance of MFG-E8 in in vivo osteoclastogenesis, we subjected WT and Mfge8^−/− mice to ligature-induced periodontitis. Mfge8^−/− mice exhibited more bone loss and increased osteoclastogenesis in the periodontal tissue than WT controls. Consistent with this, local gingival microinjection of an anti-MFG-E8 mAb (but not isotype control) exacerbated ligature-induced periodontal bone loss in WT mice. Moreover, in a model of aging-associated periodontitis, Mfge8^−/− mice experienced >60% more naturally occurring chronic periodontal bone loss than age-matched WT controls. Taken together, these data support the importance of endogenous MFG-E8 in bone homeostasis ^[10]. In this regard, our preliminary (unpublished) experiments using micro-computed tomography (μCT) indicated a modest reduction in the tissue mineral density of tibiae of Mfge8^−/− mice as compared to WT controls, suggesting that MFG-E8 might also regulate bone mass in the absence of an inflammatory condition.

MFG-E8 inhibits experimental periodontitis

The observed upregulation of MFG-E8 during osteoclastogenesis is in line with most biological systems where the expression of negative regulators is upregulated to control functional activity and prevent pathological states ^[14,15]. Whereas endogenously produced MFG-E8 acts homeostatically to restrain or mitigate unwarranted osteoclastogenesis, high concentrations of exogenously added MFG-E8 could inhibit further this process and provide a therapeutic effect. In this regard, we showed that local gingival microinjection of recombinant MFG-E8 inhibited bone loss in WT mice subjected to ligature-induced periodontitis. Similar to its effect in WT mice, recombinant MFG-E8 also protected Mfge8^−/− mice against ligature-induced bone loss. We also observed decreased expression of proinflammatory cytokines and chemokines (e.g., IL-6, IL-17, and CXCL2) in the periodontal tissue of MFG-E8 treated mice undergoing ligature-induced periodontitis (as compared with controls), which is consistent with the reported anti-inflammatory action of MFG-E8 ^[2,16,17]. The ability of MFG-E8 to inhibit the expression of proinflammatory molecules suggests an indirect way by which MFG-E8 can down-regulate osteoclastogenesis and bone loss. Taken together with the strong connection between inflammation and osteoclastogenesis ^[18], our findings suggest that the therapeutic application of MFG-E8 is capable of a two-pronged attack on periodontitis and perhaps other inflammatory bone disorders (e.g., rheumatoid arthritis and ankylosing spondylitis).

Interestingly, MFG-E8 deficiency was associated with elevated total microbiota counts in the periodontal tissue and, accordingly, treatment of WT mice with rMFG-E8 significantly decreased the bacterial load. However, MFG-E8 failed to exert direct antimicrobial activity in disk inhibition zone assays. We concluded that the suppressive effect of MFG-E8 on the microbiota is likely mediated by its capacity to inhibit inflammation and thereby to limit growth of periodontal bacteria that utilize tissue breakdown products (e.g., peptides from collagen degradation and heme-containing compounds)^[19]. In this regard, earlier work showed that local treatments with anti-inflammatory or pro-resolution agents causes a significant reduction in the total counts of periodontal bacteria in animal models of periodontitis ^[20,21].

Conclusion: MFG-E8 as a potential therapeutic in inflammatory bone loss

In contrast to inflammatory conditions, such as sepsis, colitis, acute lung injury, ischemia/reperfusion injury, atherosclerosis, and Alzheimer’s disease, where MFG-E8 expression is downregulated ^{[1,22, 23]}, there are certain pathological conditions (chronic pancreatitis, obesity, and tumorigenesis) in which MFG-E8 is expressed at high levels and is implicated in their pathogenesis ^[24–26]. Therefore, caution is required in future MFG-E8 based therapeutic strategies involving systemic administration, although the local administration of MFG-E8 in conditions with localized bone loss (e.g., periodontitis and rheumatoid arthritis) is unlikely to involve undue risks. In conclusion, our study shows that endogenously produced MFG-E8 acts in an autocrine manner to regulate OCL homeostasis, and provides proof-of-principle that recombinant MFG-E8 can be considered as a new therapeutic platform for the treatment of inflammatory bone loss.