Title: Tyrosine Kinase Pyk2 Regulates Estrogen Signaling to Control Osteoblast Mineralization
Jennifer Wu (Presenter)
Indiana University School of Dentistry
Patrick McIntyre, Indiana University
Jung Min Hong, Indiana University School of Dentistry
Sumana Posritong, Indiana University School of Dentistry
Rasika Kolte, Indiana University School of Dentistry
Angela Bruzzaniti, Indiana University
Objectives: Bone mass and regeneration is controlled by hormonal and cellular factors. The proline-rich tyrosine kinase 2 (Pyk2) is important for osteoblast (OB) activity, and Pyk2 knockout (Pyk2-KO) mice have high bone mass. We found that supplementation of ovariectomized (OVX) Pyk2-KO female mice with 17β-estradiol (E2), the major female hormones controlling bone mass, results in a greater increase in bone mass than in WT OVX mice. In the current study, we examined the role of Pyk2 and estrogen or raloxifene, a selective ER modulator, on osteoblast activity.
Methods: Calvarial OBs from Pyk2-KO and WT neonatal mice were cultured for up to 28 days in osteogenic media supplemented with 1 or 10 nM E2 or raloxifene. In addition, mineralization studies were performed in the presence of PHTPP, an antagonist of the estrogen receptor b (ERb). Quantitative analysis of mineral deposition was performed using an Alizarin Red-S elution assay.
Results: Pyk2-KO OBs exhibited increased mineralization compared to WT OBs, which was further increased in a concentration-dependent manner by E2, whereas WT OBs showed no changes. Similarly, raloxifene stimulated Pyk2-KO OB mineralization but had no effect on WT OBs. Western blot analysis of Pyk2-KO OBs revealed decreased levels of ERa, but not ERb. Therefore, we examined if E2 and raloxifene stimulate Pyk2-KO OBs predominately through ERb signaling. We found that the ERb antagonist, PHTPP, decreased in a concentration-dependent the mineralization of Pyk2-KO OBs cultured with E2 or raloxifene.
Conclusions: Pyk2-KO OBs exhibit increased mineralization compared to WT OBs, which is further increased by E2 or raloxifene. Together, our studies suggest that Pyk2-deletion promotes bone formation, and increases estrogen and raloxifene stimulation of OB mineralization, by altering ERa versus ERb signaling. Our studies provide insight into E2 signaling mechanisms, and suggest that strategies that target Pyk2 signaling may be important for bone regeneration applications.