Date of Award


Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

Mark Horowitz

Second Advisor

Melissa Kacena


A new paradigm has evolved in which multiple regulatory interactions between the skeletal and hematopoietic systems have been identified. Previous studies have demonstrated that megakaryocytes (MK) play a dual role in skeletal homeostasis by stimulating osteoblast proliferation and simultaneously inhibiting osteoclast (OC) development. Here we identify a novel regulatory pathway in which the main MK growth factor, thrombopoietin (TPO), directly regulates osteoclastogenesis. To study the role of TPO in OC development, spleen or bone marrow (BM) cells (2x106 cells/ml) or BM macrophages (BMM, 1x105 cells/ml) from C57BL/6 mice , as a source of OC precursors, were cultured with M-CSF (30 ng/ml) and RANKL (50 ng/ml) to induce OC formation. TPO (0.1-1000 ng/ml) and/or primary MK (0-0.5%), derived from C57BL/6 fetal livers, were titrated into these cultures and OC were identified as tartrate resistant acid phosphatase positive (TRAP+) giant cells with >3 nuclei. There was a significant, up to 15-fold reduction in OC formed when MK were added to all OC generating cultures, p < 0.001. Moreover, if OC generating cultures did not contain MK or MK progenitors, TPO treatment significantly enhanced OC formation up to six-fold, p < 0.01. This data demonstrates that MK are responsible for the inhibition of OC formation and that in cultures containing MK or MK progenitors such as BM or spleen cells, that TPO acts indirectly to inhibit OC formation by stimulating megakaryopoiesis, whereas in the absence of MK or MK progenitors TPO directly enhances OC formation. This conclusion is further supported by Real-Time PCR data which demonstrates that OC progenitors express c-mpl, the TPO receptor, albeit at low levels when compared to expression of c-mpl on MK. Finally, we have begun to dissect the c-mpl signaling pathway in OC progenitors. We have found that TPO induces tyrosine phosphorylation of several specific cellular proteins in the JAK/STAT pathway. Thus, TPO acts in a somewhat paradoxical manner by inhibiting OC formation through the stimulation of MK, while simultaneously playing a direct role in enhancing osteoclastogenesis.