Expression of osteoprotegerin mRNA and protein in murine megakaryocytes

Objective Osteoprotegerin (OPG) is a soluble member of the tumor necrosis factor receptor superfamily critically involved in the regulation of bone resorption. Within the bone microenvironment, OPG is abundantly produced by osteoblast/stromal cells, and its expression is regulated by transforming growth factor-β1 (TGF-β1). However, OPG expression and regulation in primary hematopoietic cells have not been fully investigated. Materials and methods Opg mRNA was studied in murine hematopoietic cells by semiquantitative reverse transcriptase-polymerase chain reaction. The OPG protein was identified by immunofluorescence labeling and secretion was assessed by enzyme-linked immunosorbent assay. Results Opg transcripts were detected in platelets, megakaryocytes (MK), monocytes, and B lymphocytes, but not in erythroblasts, neutrophils, and T lymphocytes. Mature MK and proplatelets exhibited strong immunostaining for OPG outside the storage α-granules, and secretion was detected in the conditioned medium. To analyze whether opg transcription in MK was influenced by TGF-β1, the opg/GpIIb mRNA ratio was compared in cultured MK derived from TGF-β1 null mutants and wild-type littermates without or after the addition of bioactive TGF-β1. No difference was seen, indicating that opg expression in MK was not modulated by TGF-β1. However, mRNA levels were increased when thrombopoietin was present in the culture medium, suggesting that MK maturation was correlated with enhanced opg expression. Conclusions With these results we document for the first time that murine MK and platelets express OPG. This suggests a novel role for MK in bone homeostasis, in addition to its role in vascular homeostasis.