A Novel Role for Thrombopoietin in Regulating Osteoclast Development in Humans and Mice

Abstract

Emerging data suggest that megakaryocytes (MKs) play a significant role in skeletal homeostasis. Indeed, osteosclerosis observed in several MK-related disorders may be a result of increased numbers of MKs. In support of this idea, we have previously demonstrated that MKs increase osteoblast (OB) proliferation by a direct cell-cell contact mechanism and that MKs also inhibit osteoclast (OC) formation. As MKs and OCs are derived from the same hematopoietic precursor, in these osteoclastogenesis studies we examined the role of the main MK growth factor, thrombopoietin (TPO) on OC formation and bone resorption. Here we show that TPO directly increases OC formation and differentiation in vitro. Specifically, we demonstrate the TPO receptor (c-mpl or CD110) is expressed on cells of the OC lineage, c-mpl is required for TPO to enhance OC formation in vitro, and TPO activates the MAPK, JAK/STAT, and NFκB signaling pathways, but does not activate the PI3K/AKT pathway. Further, we found TPO enhances OC resorption in CD14+CD110+ human OC progenitors derived from peripheral blood mononuclear cells (PBMCs), and further separating OC progenitors based on CD110 expression enriches for mature OC development. The regulation of OCs by TPO highlights a novel therapeutic target for bone loss diseases and may be important to consider in the numerous hematologic disorders associated with alterations in TPO/c-mpl signaling as well as in patients suffering from bone disorders.

Lnk Deficiency Leads to TPO-Mediated Osteoclastogenesis and Increased Bone Mass Phenotype

“…) and in vivo as was documented by the significant upregulation in N.Oc/T.Ar observed in Lnk −/− mice (Table ). Further, we demonstrated that stimulation of OC progenitors with TPO increased mature OC number in cultures generated from both WT and Lnk −/− mice (the former confirms our previous findings, [Bethel et al, ]). Lnk −/− OC progenitors are more responsive to TPO presumably because removal of Lnk improves TPO signaling.…”

“…In addition, MK have been implicated as secreting a, yet to be identified, factor(s) which can significantly inhibit OC development in vitro [Kacena et al, ]. Interestingly, these studies and recently published studies from our laboratory demonstrate TPO and MK opposing roles in the regulation of OC formation [Bethel et al, ].…”

C‐Mpl Is Expressed on Osteoblasts and Osteoclasts and Is Important in Regulating Skeletal Homeostasis

“…With respect to in vitro osteoclastogenesis we found OC progenitors express c-Mpl (Bethel et al,2015), WT BM cells respond to OB mediated regulation of osteoclastogenesis to a higher degree than c-Mpl−/− BM cells (Figure 3), and c-Mpl−/− OBs supported osteoclastogenesis better than WT OBs (Figure 3). Next, in an attempt to further understand how c-Mpl deficient OBs better support osteoclastogenesis, we examined OB expression of known regulators of osteoclastogenesis (Figure 4).…”

“…As shown in Figure 2A, immunoprecipitation studies demonstrated the expression of c-Mpl in MKs (positive control) and in WT OBs, but not in c-Mpl À/À OBs (negative control). With respect to expression in OC lineage cells, we recently reported that primary bone marrow macrophages (OC progenitors) as well as two OC-like cell lines (RAW264.7 and a PAX5 Spleen Cell Line generated by M. Horowitz) all expressed c-Mpl albeit at lower levels than that observed in MKs [Bethel et al, 2015].…”

“…We recently demonstrated that TPO significantly enhanced osteoclastogenesis when bone marrow marcophages (BMMs) were used as a source of OC progenitors, and that fewer OCs were generated from c-Mpl À/À BMMs as compared to those generated from WT BMMs [Bethel et al, 2015]. As fewer OCs were generated from c-Mpl À/À BMMs in vitro but higher numbers of OCs were observed in c-Mpl À/À mice in vivo, and because we found that OBs also express c-Mpl, we sought to determine whether c-Mpl À/À OBs promote osteoclastogenesis.…”