University thesis:
Dissertation, Universität Freiburg, 2018
Footnote:
Description:
Abstract: Fibrodysplasia ossificans progressiva (FOP) is a rare and highly disabling human disease characterized by extensive and progressive extraskeletal bone formation. The disease is caused by a heterozygous point mutation (R206H in classic FOP) in the activation domain of Alk2, a BMP (bone morphogenic protein) type 1 receptor. This gain-of-function mutation renders Alk2 responsive to a panel of ligands like Activin A, which are normally antagonists for wildtype receptors, resulting in excessive and uncontrolled bone growth. To date, only limited therapeutic options to counteract FOP are available. Here, I analyzed whether Pasteurella multocida toxin (PMT) is able to affect osteoblast differentiation in the context of FOP. PMT is a 146 kDa protein toxin which deamidates an essential glutamine residue in the switch II domain in the α-subunit of heterotrimeric G proteins (Gq/11, Gi and G12/13) resulting in glutamate. Due to an inhibition of the intrinsic GTPase activity in the Gα-subunit, PMT constitutively activates heterotrimeric G protein signaling. By this mechanism of action, PMT is able to profoundly modulate bone metabolism: the toxin has been shown to activate osteoclast and to inhibit osteoblast differentiation leading to the phenotype of atrophic rhinitis in pigs, a disease of bone degradation caused by PMT.<br>C2C12 myoblasts are a well-established model to study osteoblast differentiation in cell culture. Here, I report on heterotrimeric G protein deamidation and activation of downstream signaling pathways induced by PMT in C2C12 cells. I show that the toxin very potently (EC50 of 1.2 pM) inhibited BMP-4-induced osteoblast differentiation, which was determined by measuring alkaline phosphatase (ALP) activity as an early differentiation marker. On the basis of C2C12 cells, I engineered a FOP cell culture model by expressing Alk2(R206H) under the control of a doxycycline-inducible promoter. BMP-4 stimulated osteoblastogenesis both in cells overexpressing wildtype Alk2 and in C2C12-Alk2(R206H) cells. In contrast, Activin A only induced ostoblastogenesis in cells expressing the mutant receptor. Specifically, Activin A increased ALP activity up to 20-fold. Most importantly, PMT strongly inhibited differentiation induced both by BMP-4 and Activin A. An EC50 of 3.5 pM was calculated for inhibition of Activin A-induced ALP activity by PMT. Although PMT activates different heterotrimeric G protein family members, only the expression of active Gq could mimic the toxin’s effect on Activin A-induced osteoblast differentiation. Furthermore, inactivation of Gq/11 by the specific inhibitor FR900359 blocked the PMT effect. In line with that, the stimulation of canonical second messengers and effectors of Gαq inhibited Activin A-mediated formation of osteoblasts in C2C12-Alk2(R206H) cells. Both increasing intracellular Ca2+-concentrations via ionophore A23187 and activating PKC (protein kinase C) by PMA (phorbol 12-myristate 13-acetate) inhibited osteoblast differentiation. Notably, Activin A-induced osteoblastogenesis in C2C12-Alk2(R206H) cells was also inhibited by stimulation of the α1A-adrenoceptor, which couples to Gαq, by phenylephrine.<br>PMT had no effect on Alk2-induced activating phosphorylation in the SSXS motif of Smad1/5/9, which are the major downstream effectors of the Alk2 receptor. In addition, nuclear translocation of the activated Smad complex was not affected by PMT. However, PMT diminished BMP-4- and Activin A-induced gene expression, which I showed both by expression analysis of a panel of osteoblast marker genes by RT-qPCR experiments and by measuring the expression of a luciferase reporter coupled to a Smad1/5/9-responsive promoter. These results suggest that the toxin affects DNA binding of the Smad complex. <br>In summary, my data indicate that PMT potently inhibits osteoblastogenesis in a FOP cell culture model by activating Gq-Ca2+-signaling. Due to the fact that G protein-coupled receptors are highly druggable cellular targets, I suggest that activation of Gq protein-coupled receptors and of Gq-signaling might be a rationale for pharmacological therapy of FOP