Healthy bone mass in adults is primarily maintained by constant and dynamic remodeling between osteoclast-mediated bone resorption and osteoblast/osteocyte-mediated bone formation (Sims and Martin, 2014; Raggatt and Partridge, 2010). Osteoclasts are giant multinucleated cells derived from myeloid macrophage lineage that function exclusively as bone resorbing cells. Osteoblasts are derived from bone marrow mesenchymal stem cells and differentiate into mature osteoblasts lining bone surface, and osteocytes embedded in bone. These cells are responsible for extracellular matrix formation and mineralization to form bone. Crosstalk between osteoclasts and osteoblasts/osteocytes coordinately couples their activities to ensure that osteoclast-generated resorption lacunae are filled with new bone produced by osteoblasts so as to maintain bone homeostasis during bone remodeling (Sims and Martin, 2014; Raggatt and Partridge, 2010). This provides an important mechanism for adapting the skeleton to changing biomechanical influences and repairing bone damage throughout life.

The balance of this bone remodeling process, however, is disrupted in pathological conditions (Goldring et al., 2013; Schett and Gravallese, 2012; Schett and Sieper, 2009; Walsh et al., 2009). For example, excessive bone loss but limited bone formation often occur in postmenopausal osteoporosis, rheumatoid arthritis (RA), periodontitis, peri-prosthetic loosening, Paget’s disease, and osteolytic bone tumors. On the other hand, excessive bone formation and/or defective bone erosion result in osteopetrosis or osteosclerosis. The mechanisms that regulate bone remodeling in physiological and pathological conditions are complex and far from well understood. In addition to diverse coupling factors, bone remodeling is also regulated by the molecular mechanisms that simultaneously control the differentiation and activities of the major cellular participants, osteoclasts, and osteoblasts/osteocytes. These mechanisms are underappreciated but important for understanding skeletal physiology and pathology as well as providing novel strategies to target both bone cell types, thereby effectively restoring normal remodeling for healthy skeleton. Recent joint association study of a genome-wide association study (GWAS) and a meta-analysis of bone mineral density (BMD) at different skeletal sites identified Differentially Expressed in FDCP 6 homolog (DEF6) as a novel loci associated with BMD (Pei et al., 2019). This new finding brought Def6 into the spotlight. Further studies are thus needed to elucidate the role of Def6 in bone remodeling and underlying mechanisms.

Def6, also known as IRF4-binding protein (IBP) or SWAP-70-like adaptor protein of T cells (SLAT), was originally identified as an activator of Rho GTPases with unique molecular structures and domains that have limited homology with other classical Rho-family guanine nucleotide exchange factors (GEFs) (Gupta et al., 2003; Tanaka et al., 2003). Def6 has a potential calcium binding EF-hand domain on the N terminus, followed by an immunoreceptor tyrosine-based activation motif-like sequence, a PI(3,4,5)P3-binding pleckstrin-homology (PH) domain, and a Dbl-homology (DH) domain exhibiting GEF activity on the C terminus (Gupta et al., 2003; Mavrakis et al., 2004; Biswas et al., 2010). This molecular structure is different from other typical GEFs, in which the GEF domain is on the N terminus of the PH domain. This molecular structure difference may contribute to the diverse biological functions of Def6 that distinguish it from other classical GEFs. Indeed, literature has shown that Def6 functions as a multifunctional protein that plays a variety of roles in immunology through diverse downstream effectors in addition to its GEF activity (Biswas et al., 2010; Altman and Bécart, 2009). Def6 is highly expressed in T cells and plays important roles in T cell proliferation, Th1/Th2 lineage differentiation and function. For instance, Def6 regulates IL-17 and IL-21 production in T cells via binding to and preventing the transcriptional activity of IRF4 (11, 13). Def6 associates with IP3 receptor 1 to regulate calcium signaling in T cells (Fos et al., 2014). Def6 is also expressed in myeloid cells and regulates innate immunity (Chen et al., 2009). Def6 deficient mice crossed with TCR transgenic DO11.10 mice develop RA-like joint disease with bone erosion (Chen et al., 2008). Both genetic mouse models and recent evidence obtained from patients (Serwas et al., 2019; Sun et al., 2016; Yi et al., 2017; Manni et al., 2018) show a critical role for Def6 in autoimmunity.

In addition to the immunoregulatory function of Def6, we have identified Def6 as a novel inhibitor of osteoclastogenesis in both physiological and inflammatory conditions (Binder et al., 2017). In RA patients, the serum TNF-α levels and disease activity are closely correlated with Def6 expression in osteoclast precursors. TNF-α downregulates Def6 expression in osteoclasts. Blocking the activity of TNF-α by TNF inhibitor treatment significantly increases Def6 expression levels, meanwhile decreasing osteoclast differentiation in RA osteoclast precursors, which supports an inhibitory role for Def6 in TNF-α induced osteoclastogenesis in humans. However, the role of Def6 in osteoblasts and bone formation has not been explored.

In the present study, we found that Def6 suppresses osteoblast differentiation and mineralization both in vitro and in vivo. Def6 knockout (KO) mice exhibit osteoporotic phenotype with enhanced osteoclast formation. Osteoblast differentiation and bone formation are elevated as well in Def6 KO mice, indicating a high bone turnover rate that leads to bone loss in Def6 KO mice. Thus, lack of Def6 leads towards unbalanced activities between osteoclastic resorption and osteoblast-mediated bone formation, disrupting normal bone remodeling. Recent studies from the osteoimmunology field highlight the critical role for the immune system in the regulation of bone homeostasis, mostly on bone resorbing osteoclasts (Takayanagi, 2015; Goldring, 2013; Lorenzo and Choi, 2005; Schett, 2009; Pacifici, 2013). In this study, we found that Def6 suppresses osteoblast differentiation via endogenous type-I IFN response-mediated feedback inhibition, pointing to important immunoregulation of osteoblasts and bone formation. These findings reveal that Def6 is a novel bone remodeling regulator in osteoimmunology that controls both osteoclast and osteoblast differentiation through immunoregulatory mechanisms to maintain bone remodeling.

The adult skeleton undergoes constant and dynamic remodeling process throughout life to maintain bone homeostasis, which is achieved by the tight control of coupling between osteoclast-mediated bone resorption and osteoblast-mediated bone formation (Sims and Martin, 2014; Raggatt and Partridge, 2010). The studies of cell differentiation and function for each cell type are extensive. However, the mechanisms that simultaneously regulate both cell types during bone remodeling are less appreciated. Understanding of these mechanisms can help identify novel therapeutic targets for regulating the activity of both cell types involved in bone remodeling, and thereby facilitating and enhancing treatment efficacy. Our previous work identified Def6 as an important inhibitory regulator for osteoclastogenesis and bone resorption. In this study, we found that Def6 is an inhibitor that suppresses osteoblastic differentiation and osteogenesis in vivo. Therefore, Def6 is a common regulator that negatively regulates both osteoclastogenesis and osteoblastogenesis simultaneously in bone remodeling. Def6 deficiency enhances both osteoclastic bone resorption and osteoblastic bone formation. However, the regulatory potency by Def6 in the two cell types is unbalanced, leading to a high turnover osteoporotic phenotype in Def6^-/- mice. Hence, Def6 is an important regulator that maintains physiological bone homeostasis. Environmental cues or pathological conditions that affect Def6 expression and activity may deregulate bone remodeling and impact bone mass.

In this study, we utilized a well-established and widely used in vitro osteoblast culture system, in which the osteoblastic cells were isolated from the calvaria of newborn mice. Since Def6 plays an inhibitory role in macrophage differentiation into osteoclasts, we validated whether the calvarial osteoblast culture system is solid to investigate the role of Def6 in osteoblastogenesis without significant macrophage lineage effects. Using RNAseq, a sensitive approach, we can only detect very low expression levels (below 1–2 counts per million reads) of marker genes for either macrophages or osteoclasts in this culture system. Furthermore, these genes belong to non-significant genes and there are no differences in their expression between WT and Def6 deficient osteoblast cultures (Figure 1—figure supplement 2). To exclude any potential effect from hematopoietic cells, we removed CD45 positive cells, which label all hematopoietic cells, and selected CD45 negative calvarial osteoblasts to compare the osteoblast differentiation. Similarly as the results in non-select cultures shown in Figures 1 and 3, Def6 deficiency significantly enhanced osteoblastogenesis and osteoblast marker gene expression, but reduced ISG gene expression without CD45 positive cells in cultures (Figure 1—figure supplement 3). Moreover, we co-cultured CD45 negative calvarial osteoblasts with bone marrow derived macrophages (BMMs) isolated from WT or Def6 deficient mice. We found that Def6 deficiency in BMMs does not affect osteoblast differentiation (Figure 1—figure supplement 4). Therefore, these results collectively validated this well-established osteoblast culture system, which is solid to test Def6 function in osteoblast differentiation.

Accumulating evidence shows that Def6 acts as a multifunctional protein through its GEF activity or binding to other regulators, such as IRF4 and IP3 receptor 1, to regulate the activities of immune cells including T cells and myeloid cells. A large body of work has also established the immunoregulatory function of Def6 in innate and adaptive immunity as well as in autoimmunity. The studies (Binder et al., 2017, and current study) from our group revealed an important novel role for Def6 in maintaining bone homeostasis, in which Def6 functions as an inhibitor that suppresses the differentiation of both osteoclasts and osteoblasts. Interestingly, we found that Def6 executes this inhibitory function via endogenous type-I IFN-mediated feedback inhibition of bone cell differentiation, the same downstream mechanisms by which Def6 suppresses differentiation of both osteoclasts and osteoblasts. In the osteoclast differentiation process, it is well documented that RANKL or TNFα treatment induces type-I IFN expression in macrophages/osteoclast precursors. Although the magnitude of type-I IFN induction by RANKL or TNFα is small (low picomolar concentrations) when compared with other stimuli such as toll-like receptor (TLR) stimulation or viral infection, the endogenous autocrine type-I IFNs show significant feedback inhibitory effects on osteoclast differentiation (Binder et al., 2017; Takayanagi et al., 2002; Inoue et al., 2018). Our group and others have shown that activation of type-I IFN signaling by exogenous IFNβ suppresses osteoblast differentiation, matrix formation and mineralization (Woeckel et al., 2012a; Woeckel et al., 2012b; Kim et al., 2003). However, the effects of endogenous or basal IFN activity in osteoblastic cells on their differentiation have recently drawn attention with variable reports from literature (Takayanagi et al., 2002; Kota et al., 2018). We and other groups (Kota et al., 2018) recently detected endogenous ISG expression in osteoblastic culture system in the absence of virus, pathogens or exogenous IFNβ. Interestingly, ISG gene expression is induced along osteoblast differentiation, but is reversely correlated with the osteogenic potential between different cultures, for example, Def6^-/- osteoblastic cells express lower ISGs but have higher osteogenic potential than WT cells. The puzzle is what is the inducer of the ISG expression in osteoblastic cells. When we added IFNβ blocking antibody to the cultures to block potential IFNβ, we found that the ISG expression was largely decreased, indicating that the osteoblastic cells secrete IFNβ, which subsequently induces ISG expression. Indeed, our ELISA results verified autocrine IFNβ, but not IFNα, in the osteoblast cultures. Despite that the amount of autocrine IFNβ is low (below 5 pg/ml), its biological function in ISG induction and osteoblastic inhibition is clearly present. This is not surprising for homeostatic conditions (Gough et al., 2012) or certain autoimmune diseases, such as SLE, in which an 'IFN signature' of ISG expression is observed although IFNβ may not be detectable (Ivashkiv and Donlin, 2014). Studies reveal the existence of constitutive type-I IFN in healthy mice that are maintained in specific pathogen-free environments. Constitutive type-I IFNs are usually produced at vanishingly low amounts in the absence of infection and yet have a variety of biological effects, such as maintenance of the hematopoietic stem cell (HSC) niche, regulation of immune cell function and osteoclastogenesis (Gough et al., 2012). These highlight the important biological roles for endogenous IFN in cells/tissues. Since the expression levels of endogenous type-I IFN are usually low, close to the threshold of detection, the IFN activity is also commonly indicated and reflected by the ISG expression in these conditions. Our results thus unveil an important novel immunoregulatory function of Def6 in bone cells, in which Def6 acts as an upstream regulator of IFNβ and ISG gene expression and endogenous type-I IFN response to regulate bone cell differentiation. These findings highlight the biological importance of the close interaction of immune system and bone health.

Induction of Ifnb1 expression involves, in most cases, the activation of AP-1, NF-κB and various interferon regulatory factors (IRFs), depending on the cell types and context of the stimulation (Pitha and Kunzi, 2007). In response to strong IFN inducers, such as virus or TLR stimulation, Ifnb1 induction often occurs rapidly and strongly within minutes to a few hours and is dependent on IRF3/7 (Pitha and Kunzi, 2007). In contrast, Ifnb1 induction in osteoclast or osteoblast cultures occurs slowly and mildly. TNFα–induced IFNβ expression in monocytes/macrophages is mainly dependent on IRF1 (Yarilina et al., 2008) and Def6 (Binder et al., 2017). RANKL induces IFNβ expression via c-Fos and Def6 during osteoclastogenesis (Binder et al., 2017; Takayanagi et al., 2002). This RANKL-induced autocrine IFNβ expression is negatively controlled by miR182-PKR axis (Inoue et al., 2018). The attention on the endogenous autocrine IFNβ loop is just emerging. How IFNβ is induced in osteoblastic cells was unknown. We, for the first time, identified Def6 as an upstream regulator that modulates endogenous IFNβ expression and activity, significantly via PKR, during osteoblastogenesis. PKR was originally identified as an IFNβ inducible gene in response to viral infection, and plays an important role in type-I IFN production (Haller et al., 2006; Munir and Berg, 2013; Meurs et al., 1990; McAllister et al., 2012; Taghavi and Samuel, 2012). Various downstream pathways, such as IFNβ and eIF2a-mediated translational inhibition, can be regulated by PKR-dependent on cell types and context (Yang et al., 2010; Opitz et al., 2009; Lourenco et al., 2013; Hsu et al., 2004). In osteoblasts, we did not observe phosphorylation of eIF2α (data not shown). Instead, IFNβ was identified as a significant downstream factor mediated by PKR in osteoblasts, corroborated by our data that PKR deficiency almost abrogates the expression of IFNβ and downstream ISG genes. On the other hand, PKR, as an IFNβ inducible gene, could also be regulated by the autocrine IFNβ in the osteoblast cultures, leading to a regulatory circuit between PKR and IFNβ downstream of Def6 that coordinately mediate osteoblastogenesis. In addition, Stat1 is an ISG gene and previous work demonstrates its inhibitory function in osteoblast differentiation by binding to and preventing the activity of Runx2 (Kim et al., 2003). A recent study shows that Gbp1, an ISG gene, inhibits osteogenic differentiation (Bai et al., 2018). It seems that the ISG genes play important roles in osteoblastic inhibition, which is distinct from their originally identified anti-viral function. Def6 deficiency significantly decreases a group of ISG gene expression, including Stat1 and Gbp3, which belongs to Gbp family. Thus, Def6 may also function through these ISGs to suppress osteogenesis. It would be of great interest for future studies to fully dissect the underlying molecular mechanisms by which PKR and other ISGs regulate osteoblast differentiation and bone formation.

Recent human genetic evidence, together with results obtained from animal models and patients, reveal the important regulatory role for Def6 in immunity and autoimmune diseases, such as SLE and RA. Def6 deficient female mice on a mixed 129/BL6 background develop a lupus-like syndrome (Biswas et al., 2010; Fanzo et al., 2006). Def6 is a new systemic lupus erythematosus (SLE) risk variant (Sun et al., 2016) and recent evidence obtained from patients show a critical role for Def6 in autoimmunity (Serwas et al., 2019). In addition to the role for Def6 in immune diseases, recent GWAS study identifies DEF6 as a novel loci associated with BMD (Pei et al., 2019). Along this line, our results show that Def6 is a key bone remodeling regulator that regulates both osteoclasts and osteoblasts to maintain bone homeostasis. In disease settings, Def6 deficient DO11.10 mice on the Balb/c background with TCR activation developed a RA-like joint disease with bone erosion (Chen et al., 2008). In our previous study, we uncovered a key role of Def6 in inhibiting TNF-induced osteoclast formation and inflammatory bone resorption, and importantly, we provided evidence demonstrating a significant reverse correlation between Def6 expression levels and TNF-α activity in RA osteoclast precursors (Binder et al., 2017). Thus, our results support an important role for Def6 in human RA disease. In addition to chronic inflammation, the bone remodeling in RA is deregulated, featured by enhanced bone resorption but drastically reduced new bone formation, which is a long-standing challenge for treating bone damage in RA. We found that TNFα drastically decreases Def6 expression in osteoclast differentiation, and the decreased Def6 in turn promotes osteoclastogenesis and bone resorption. However, TNFα does not significantly affect Def6 expression in osteoblasts (Figure 1—figure supplement 5). Thus, Def6 maintains its inhibitory role in osteogenesis in this inflammatory setting. The differential regulation of Def6 expression by TNFα in osteoclasts and osteoblasts may contribute to enhanced bone resorption and reduced bone formation in the presence of TNFα. Taken together, the Def6-IFNβ axis plays a key role in regulating the differentiation of both osteoclasts and osteoblasts to maintain bone homeostasis, and targeting Def6 in bone cells might represent an effective strategy to control inflammatory bone resorption.

RNA-seq data (accession #GSE148455) have been deposited in NCBI's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE148455).

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   Def6 regulates endogenous type I interferon responses in osteoblasts and suppresses osteogenesis.

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Author details

1. Zhonghao Deng

   Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Courtney Ng and Kazuki Inoue

   Competing interests

   No competing interests declared

2. Courtney Ng

   Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, United States

   Contribution

   Formal analysis, Validation, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Zhonghao Deng and Kazuki Inoue

   Competing interests

   No competing interests declared

3. Kazuki Inoue

   1. Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, United States
   2. Department of Medicine, Weill Cornell Medical College, New York, United States

   Contribution

   Software, Formal analysis, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Zhonghao Deng and Courtney Ng

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6305-9374

4. Ziyu Chen

   Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

5. Yuhan Xia

   Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, United States

   Contribution

   Investigation, Visualization

   Competing interests

   No competing interests declared

6. Xiaoyu Hu

   Institute for Immunology and School of Medicine, Tsinghua University, Beijing, China

   Contribution

   Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

7. Matthew Greenblatt

   1. Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, United States
   2. Research Division, Hospital for Special Surgery, New York, United States

   Contribution

   Resources, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

8. Alessandra Pernis

   1. Autoimmunity and Inflammation Program, Hospital for Special Surgery, New York, United States
   2. Graduate Program in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, New York, United States

   Contribution

   Resources, Visualization, Writing - review and editing

   Competing interests

   No competing interests declared

9. Baohong Zhao

   1. Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, United States
   2. Department of Medicine, Weill Cornell Medical College, New York, United States
   3. Graduate Program in Cell and Development Biology, Weill Cornell Graduate School of Medical Sciences, New York, United States

   Contribution

   Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   zhaob@hss.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1286-0919

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