Pasteurella Multocida Toxin Prevents Osteoblast Differentiation by Transactivation of the MAP-Kinase Cascade via the Gα _q/11 - p63RhoGEF - RhoA Axis

Abstract

The 146-kDa Pasteurella multocida toxin (PMT) is the main virulence factor to induce P. multocida-associated progressive atrophic rhinitis in various animals. PMT leads to a destruction of nasal turbinate bones implicating an effect of the toxin on osteoblasts and/or osteoclasts. The toxin induces constitutive activation of Gα proteins of the G _q/11-, G _12/13- and G _i-family by deamidating an essential glutamine residue. To study the PMT effect on bone cells, we used primary osteoblasts derived from rat calvariae and stromal ST-2 cells as differentiation model. As marker of functional osteoblasts the expression and activity of alkaline phosphatase, formation of mineralization nodules or expression of specific transcription factors as osterix was determined. Here, we show that the toxin inhibits differentiation and/or function of osteoblasts by activation of Gα _q/11. Subsequently, Gα _q/11 activates RhoA via p63RhoGEF, which specifically interacts with Gα _q/11 but not with other G proteins like Gα _12/13 and Gα _i. Activated RhoA transactivates the mitogen-activated protein (MAP) kinase cascade via Rho kinase, involving Ras, MEK and ERK, resulting in inhibition of osteoblast differentiation. PMT-induced inhibition of differentiation was selective for the osteoblast lineage as adipocyte-like differentiation of ST-2 cells was not hampered. The present work provides novel insights, how the bacterial toxin PMT can control osteoblastic development by activating heterotrimeric G proteins of the Gα _q/11-family and is a molecular pathogenetic basis for understanding the role of the toxin in bone loss during progressive atrophic rhinitis induced by Pasteurella multocida.

Author Summary

Pasteurella multocida causes as a facultative pathogen various diseases in men and animals. One induced syndrome is atrophic rhinitis, which is a form of osteopenia, mainly characterized by facial distortion due to degradation of nasal turbinate bones. Strains, which especially affect bone tissue, produce the protein toxin P. multocida toxin (PMT). Importantly, PMT alone is capable to induce all symptoms of atrophic rhinitis. To cause osteopenia PMT influences the development and/or activity of specialized bone cells like osteoblasts and osteoclasts. Recently, we could identify the molecular mechanism of PMT. The toxin constitutively activates certain heterotrimeric G proteins by deamidation. Here, we studied the effect of PMT on the differentiation of osteoblasts. We demonstrate the direct action of PMT on osteoblasts and osteoblast-like cells and as a consequence inhibition of osteoblastic differentiation. Moreover, we revealed the underlying signal transduction pathway to impair proper osteoblast development. We show that PMT activates small GTPases in a Gα _q/11 dependent manner via a non-ubiquitously expressed RhoGEF. In turn the mitogen-activated protein kinase pathway is transactivated leading to inhibition of osteoblastogenesis. Our findings present a mechanism how PMT hijacks host cell signaling pathways to hinder osteoblast development, which contributes to the syndrome of atrophic rhinitis.