An essential role for the association of CD47 to SHPS-1 in skeletal remodeling

Abstract

Integrin associated protein (IAP/CD47) has been implicated in macrophage-macrophage fusion. To understand the actions of CD47 on skeletal remodeling, we compared Cd47−/− with Cd47+/+ controls. Cd47−/− mice weighed less and had decreased areal bone mineral density compared to controls. Cd47−/− femurs were shorter in length with thinner cortices and exhibited lower trabecular bone volume due to decreased trabecular number and thickness. Histomorphometry revealed reduced bone formation and mineral apposition rates, accompanied by decreased osteoblast numbers. No differences in osteoclast number were observed despite a non-significant, but 40% decrease, in eroded surface/bone surface in Cd47−/− mice. In vitro the number of functional osteoclasts formed by differentiating Cd47−/− bone marrow cells was significantly decreased compared to wild-type cultures and was associated with a decrease in bone resorption capacity. Furthermore, by disrupting the CD47-SHPS-1 association we found that osteoclastogenesis was markedly impaired. Assays for markers of osteoclast maturation suggested the defect was at the point of fusion and not differentiation, and was associated with a lack of SHPS-1 phosphorylation, SHP-1 phosphatase recruitment and subsequent dephosphorylation of non-muscle cell myosin IIA. We also demonstrated a significant decrease in osteoblastogenesis in bone marrow stromal cells derived from Cd47−/− mice. Our finding of cell autonomous defects in Cd47−/− osteoblast and osteoclast differentiation coupled with the pronounced skeletal phenotype of Cd47−/− mice support the conclusion that CD47 plays an important role in regulating skeletal acquisition and maintenance through its actions on both bone formation and resorption.

Osteoclast Fusion: Time‐Lapse Reveals Involvement of CD47 and Syncytin‐1 at Different Stages of Nuclearity

“…Inhibition of CD47 resulted in: (i) a significant drop in the number of fusion events in real‐time (Fig. 1) in line with prior proposals (Lundberg et al, 2007; Maile et al, 2011); (ii) a strong reduction in fusion events between two mono‐nucleated preOCs; (iii) a specific inhibition of fusion between a preOCs and an OC with two nuclei, but without affecting fusion with OCs containing three nuclei or more; (iv) no effect on fusion between two multi‐nucleated OCs; (v) a specific reduction in fusion events mediated through the broad contact surface phenotype. These data fit the view discussed by Vignery (2005) that CD47 is involved in recognition of “self” and as a possible fusion partner.…”

“…Inhibition or knock‐out of CD47 was previously reported to inhibit fusion of murine OCs (Lundberg et al, 2007; Maile et al, 2011) as well as macrophages (Han et al, 2000) in cell culture. In these studies, the total number of OCs with three nuclei or more was analyzed at the end of the culture, but without reporting details on the actual number of nuclei per cell (Lundberg et al, 2007; Maile et al, 2011; Koskinen et al, 2013).…”

“…In these studies, the total number of OCs with three nuclei or more was analyzed at the end of the culture, but without reporting details on the actual number of nuclei per cell (Lundberg et al, 2007; Maile et al, 2011; Koskinen et al, 2013). In our recent study (Hobolt‐Pedersen et al, 2014), we used a CD47 blocking‐antibody and evaluated at the end of the culture the number of nuclei in all OCs both in control condition as well as in the presence of the blocking antibody.…”

“…The most prominent of these is DC‐STAMP (dendritic cell‐specific transmembrane protein) (Kukita et al, 2004; Yagi et al, 2005; Iwasaki et al, 2008; Mensah et al, 2010; Chiu et al, 2012; Chiu and Ritchlin, 2016), which has been identified as one of the most essential single factors supporting both differentiation and fusion. However, also other factors such as CD47 (Han et al, 2000; Lundberg et al, 2007; Maile et al, 2011; Koskinen et al, 2013; Hobolt‐Pedersen et al, 2014), syncytin‐1 (Soe et al, 2011), OC‐STAMP (osteoclast stimulatory transmembrane protein) (Miyamoto et al, 2012; Witwicka et al, 2015), dynamin (Shin et al, 2014; Verma et al, 2014), Pin1 (peptidyl‐prolyl cis‐trans isomerase NIMA‐interacting 1) (Islam et al, 2014; Cho et al, 2015), and e‐cadherin (Mbalaviele et al, 1995; Fiorino and Harrison, 2016) are involved in OC fusion, but it is important to stress that this list is not exhaustive. In order to identify the role of these factors , a series of molecular techniques, and cellular model systems have been employed, which in general are evaluated through end‐point measurements by counting the number of multi‐nucleated OCs, number of nuclei per OC, resorptive activity, and so forth at the end of the incubation period.…”

Lack of CD47 Impairs Bone Cell Differentiation and Results in an Osteopenic Phenotype in Vivo due to Impaired Signal Regulatory Protein α (SIRPα) Signaling

“…These findings have been confirmed in vitro both by Uluçkan et al (13) and Maile et al (14). However, Maile et al did not find any reduction in number of osteoclasts in vivo (14). To determine the role of CD47 in bone homeostasis, both in vitro and in vivo, we here investigated whether CD47 is involved in osteoclast and stromal cell/osteoblast differentiation in murine bone marrow cultures.…”