Pyk2 regulates megakaryocyte-induced increases in osteoblast number and bone formation

Abstract

Pre-clinical and clinical evidence from megakaryocyte (MK) related diseases suggest that MKs play a significant role in maintaining bone homeostasis. Findings from our laboratories reveal that MKs significantly increase osteoblast (OB) number through direct MK-OB contact and the activation of integrins. We therefore examined the role of Pyk2, a tyrosine kinase known to be regulated downstream of integrins, in the MK-mediated enhancement of OBs. When OBs were co-cultured with MKs, total Pyk2 levels in OBs were significantly enhanced primarily due to increased Pyk2 gene transcription. Additionally, p53 and Mdm2 were both decreased in OBs upon MK stimulation, which would be permissive of cell cycle entry. We then demonstrated that OB number was markedly reduced when Pyk2−/− OBs, as opposed to wild-type (WT) OBs, were co-cultured with MKs. We also determined that MKs inhibit OB differentiation in the presence and absence of Pyk2 expression. Finally, given that MK replete spleen cells from GATA-1 deficient mice can robustly stimulate OB proliferation and bone formation in WT mice, we adoptively transferred spleen cells from these mice into Pyk2−/− recipient mice. Importantly, GATA-1 deficient spleen cells failed to stimulate an increase in bone formation in Pyk2−/− mice, suggesting in vivo the important role of Pyk2 in the MK-induced increase in bone volume. Further understanding of the signaling pathways involved in the MK-mediated enhancement of OB number and bone formation will facilitate the development of novel anabolic therapies to treat bone loss diseases.

Signaling Pathways Involved in Megakaryocyte‐Mediated Proliferation of Osteoblast Lineage Cells

“…NF-E2 deficient mice have approximately a 5-fold increase in immature MK number, 5% of the normal numbers of platelets, and 2–3-fold increase in bone mass (Shivdasani, et al, 1995, Kacena, et al, 2004, Kacena, et al, 2005). This same phenomena was also recently reported, whereby spleen cells from GATA-1 deficient mice were transplanted into wild-type mice and a high bone mass phenotype was observed (Cheng, et al, 2013). In each of these models, both the hematologic phenotype and the high bone mass phenotype were adoptively transferred, suggesting a role for hematopoietic cells in this mechanism, most likely MKs (Kacena, et al, 2005).…”

“…Surviving host MKs migrate to endosteal surfaces in bone where they stimulate a 2-fold increase in OB number thus augmenting the so-called endosteal hematopoietic stem cell niches. Contact between MKs and OBs and/or their precursors have been described (Cheng, et al, 2000, Miao, et al, 2004, Kacena, et al, 2004, Ciovacco, et al, 2009, Ciovacco, et al, 2010, Lemieux, et al, 2010, Dominici, et al, 2009, Kacena, et al, 2012, Cheng, et al, 2013). As an example, Cheng et al (Cheng, et al, 2000) observed that when isolating bone marrow stromal cells (BMSCs), complexes existed consisting of BMSCs and MKs, demonstrating a physical association between these cells.…”

Osteomacs interact with megakaryocytes and osteoblasts to regulate murine hematopoietic stem cell function

“…CD45 1 cells in NCCs expressed some classical macrophage markers, but were mostly negative for others ( Figure 1A, NCC). Because megakaryocytes promote in vitro and in vivo osteoblast expansion, 7,9,25 we examined the impact of megakaryocyte stimulation on NCCs. When cultured alone for 7 days, NCCs increased in number, but the percentage of CD45 1 cells remained relatively unchanged or declined ( Figure 1A; cultured NCC, left dot plot).…”

“…3,4 We also showed that this activity is inversely correlated with osteoblast maturation and the level of CD166 expression 4,5 which is a functional marker of murine and human HSCs and niche competency. 6 Our group also demonstrated that megakaryocytes can increase osteoblast proliferation in vitro and in vivo, [7][8][9] thus implicating megakaryocytes, albeit indirectly, in HSC maintenance. These observations identified critical interactions between osteoblasts and megakaryocytes and offered a unique experimental model to assess how megakaryocytes impact HSC function via osteoblasts.…”

The secret life of a megakaryocyte: emerging roles in bone marrow homeostasis control

“…To this regard, the fibronectin integrins α4β1 and α5β1 and the glycoprotein GPIIb (CD41) were demonstrated to play a critical role in Mk-induced OB proliferation, as assessed by neutralizing antibody studies (62). Recently, Cheng et al demonstrated the importance of the non receptor tyrosine kinase Pyk2, a downstream mediator of integrin signalling, in Mk-induced OB proliferation (63). Although the increase in Pyk2 expression, obtained co-culturing Mks and OBs, was not related to β1 integrin activation, this up-regulation resulted critical for Mk-mediated OB expansion, both in vitro and in vivo , as demonstrated by the reduced responsiveness to Mk stimulation of Pyk2 −/− OBs with respect to wild type.…”