A growing body of evidence suggests that megakaryocytes (MKs) play a key role in regulating skeletal homeostasis. In support of this, mice deficient in GATA-1 or NF-E2, transcription factors required for normal MK development, exhibit an increase in immature MKs as well as a dramatic decrease in platelet numbers. Importantly, these mice exhibit a 300% increase in trabecular bone mineral density. The cellular and tissue-level mechanisms underlying this increase in bone mass remain unclear. However, our histological evaluation of GATA-1 and NF-E2 deficient mice reveals higher numbers of osteoblasts (OBs) on trabecular surfaces. Importantly, the increased bone phenotype can be adoptively transferred into irradiated wild-type mice using spleen cells from mutant mice suggesting a role for hematopoietic cells, most likely MKs which are elevated in these mice, in this mechanism. Consistent with these in vivo experiments, our in vitro data show that MKs enhance OB proliferation (up to 6-fold) by direct cell-to-cell contact which involves integrin engagement. Although the exact mechanisms by which MKs enhance OB proliferation remain to be determined, these observations suggest that the interaction of MKs with OBs results in increased osteogenesis. Furthermore, in OBs co-cultured with MKs, the expression of the cell cycle arrest protein Retinoblastoma (Rb), and murine double minute-2 (Mdm2), an E3 ubiquitin ligase that regulates proteosome mediated degradation, are transiently decreased. Recently, we discovered that MKs regulate the temporal expression of two isoforms of the proline-rich tyrosine kinase 2 (Pyk2), a key protein kinase involved in signaling downstream of activated integrins, and that Pyk2 forms a complex with Rb and Mdm2. Moreover, the MK-mediated increase in OB number was abolished in OBs from Pyk2-/- mice. Therefore, our central hypothesis is that MKs have a anabolic effect on bone by regulating cell cycle progression in OBs via a pathway involving the Pyk2-mediated regulation of upstream and downstream signaling proteins.
In Aim 1 we will demonstrate the functional role of Pyk2 isoforms in MK-regulated OBs by ectopically expressing either Pyk2 or Pyk2-S in OBs and assessing cell cycle regulation and differentiation.
In Aim 2 we will determine the role of Pyk2's phosphorylation and activity in regulating its interaction with Rb and Mdm2 and its degradation. Finally, in Aim 3 we will demonstrate the role of Pyk2 in MK-induced bone formation by transplanting Pyk2-/- mice with hematopoietic precursors enriched with MKs. Successful accomplishment of these Aims will demonstrate the importance of MKs in regulating anabolic bone formation and the role of Pyk2 in OB cell cycle regulation. Identifying the pathways that lead to enhanced bone volume in vivo will lead to the development of novel therapeutic approaches that stimulate bone formation for the treatment of osteoporosis and other bone loss diseases.

Public Health Relevance

Megakaryocytes, the platelet producing cells, can increase bone mass in mice and humans. We have found that megakaryocytes increase osteoblast proliferation (the cells that form bone) and are trying to understand how this occurs. We hope that this work will eventually offer new strategies to treat bone loss diseases such as osteoporosis.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AR060332-04
Application #
8709999
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Sharrock, William J
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Orthopedics
Type
Schools of Medicine
DUNS #
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
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Eleniste, Pierre P; Huang, Su; Wayakanon, Kornchanok et al. (2014) Osteoblast differentiation and migration are regulated by dynamin GTPase activity. Int J Biochem Cell Biol 46:9-18
Kacena, Melissa A; Gundberg, Caren M; Kacena 3rd, William J et al. (2013) The effects of GATA-1 and NF-E2 deficiency on bone biomechanical, biochemical, and mineral properties. J Cell Physiol 228:1594-600
Eleniste, Pierre P; Du, Liping; Shivanna, Mahesh et al. (2012) Dynamin and PTP-PEST cooperatively regulate Pyk2 dephosphorylation in osteoclasts. Int J Biochem Cell Biol 44:790-800
Kacena, Melissa A; Eleniste, Pierre P; Cheng, Ying-Hua et al. (2012) Megakaryocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts. J Biol Chem 287:17257-68