JunB as a Downstream Mediator of PTHrP Actions in Cementoblasts

Abstract

The role of AP-1 family members in the action of PTHrP was examined in cementoblasts. PTHrP increased mRNA and protein levels of all Fos members, but only one Jun member (JunB) was increased. Overexpression of JunB in cementoblasts mimicked actions of PTHrP to support osteoclastogenesis and inhibit cementoblast differentiation, suggesting that the actions of PTHrP on mesenchymal cells operate through JunB. Cementoblasts are mesenchymal cells that share phenotypic features with osteoblasts in vitro; however, unlike osteoblasts, cementoblasts rarely support osteoclastogenesis in vivo. The osteoblast-mediated support of osteoclastogenesis involves PTH-induced reduction in osteoprotegerin (OPG) expression. PTH acts on osteoblastic cells through specific signaling pathways and transcription factors such as activator protein 1 (AP-1). The purpose of this study was to determine the impact of PTH-related protein (PTHrP) on AP-1 transcription factors in cementoblasts and the role of JunB in the actions of PTHrP. Cementoblastic cells were treated with PTHrP and evaluated for mRNA and protein levels of AP-1 family members. Stable transfectants of OCCM cells overexpressing JunB were evaluated for OPG production, ability to support osteoclastogenesis, and measures of proliferation and differentiation. PTHrP treatment in vitro resulted in a time-dependent upregulation of mRNA and proteins for the Fos family members, but only JunB of the Jun family. OPG mRNA and protein levels were reduced by PTHrP in OCCM and were lower in JunB overexpressing cells than controls. In co-culture experiments, TRACP+ cells were increased with RANKL treatment in JunB overexpressing cells compared with controls. Cementoblast differentiation was reduced with overexpression of JunB as measured by a decrease in mineralized nodule formation and gene expression for bone sialoprotein and osterix. Measures of proliferation including cell number and cyclin D1 levels were increased in JunB overexpressing clones. In vivo, cementoblast implants exhibited a cementoblastoid nature with copious mineral-like matrix, whereas JunB-overexpressing implants were densely cellular with little mineralized matrix. JunB was the only Jun family member increased by PTHrP, and its overexpression showed similar patterns of gene expression and OPG production as PTHrP treatment of controls. These data suggest that JunB may be a key mediator of PTHrP actions in cementoblasts.