Rho and Rho kinase are involved in parathyroid hormone-stimulated protein kinase C α translocation and IL-6 promoter activity in osteoblastic cells

Abstract

The role of small G-proteins in PTH-stimulated PKC translocation and IL-6 promoter expression in UMR-106 cells was determined. The effects of PTH(1-34) and PTH(3-34) in stimulating PKCα translocation and IL-6 were inhibited by agents that interfere with the activity of small G-proteins of the Rho family and with the downstream kinase Rho kinase. Introduction: Activation of protein kinase C (PKC) is a signaling mechanism by which parathyroid hormone (PTH) modulates interleukin-6 (IL-6) in osteoblasts, leading to osteoclastogenesis and bone resorption. PKCα and PKCβI are translocated after treatment with PTH in UMR-106 osteoblastic cells; however, the pathway leading to PKC isozyme translocation is not established. Diacylglycerol (DAG) generation from phospholipase D (PLD) is one pathway of PKC activation, and PTH-mediated PLD activity is dependent on small G-proteins of the Rho family. This study investigated whether Rho proteins modulate the PKCα translocation and IL-6 promoter activity stimulated by PTH in UMR-106 cells. Materials and Methods: UMR-106 cells were treated with PTH(1-34) or PTH(3-34). PKC translocation was determined by immunofluorescence, Rho A activation by Rhotekin assay and by translocation assessed by Western blotting in membrane and cytosol fractions, and IL-6 promoter expression by luciferase assay. Results and Conclusions: Inhibition of Rho proteins with Clostridium difficile toxin B or inhibition of Rho prenylation with GGTI attenuated PTH(1-34)- and PTH(3-34)-stimulated translocation of endogenous PKCα and IL-6 promoter activity. Expression of a constitutively active RhoA (RhoA63L) mimicked the effect of PTH(1-34) or PTH(3-34) to promote membrane localization of PKCα, whereas cells expressing a dominant negative RhoA (RhoA19N) did not respond to PTH(1-34) or PTH(3-34). The Rho kinase inhibitor Y27632 attenuated PTH(1-34)-and PTH(3-34)-stimulated PKCα translocation and IL-6 promoter activation. Rho seemed to be acting at a step before production of diacylglycerol (DAG), because the stimulation of PKCα translocation by the DAG mimetic phorbol 12,13 dibutyrate (PDBu) was unaffected by C. difficile toxin B or Y27632. These results indicate that Rho proteins are an important component of PTH signaling in osteoblastic cells and provide further demonstration of convergence between PKC and small G-protein signaling pathways. © 2004 American Society for Bone and Mineral Research.