RESUMEN
The human disease fibrodysplasia ossificans progressiva (FOP) is a rare and highly disabling disorder of extensive heterotopic bone growth that is caused by a point mutation (R206H) in the activation domain of Alk2, a BMP (bone morphogenic protein) type 1 receptor. The mutation leads to extensive BMP-signaling induced by Activin A, which is normally an antagonist for wildtype receptors, resulting in excessive and uncontrolled bone formation. Here, we studied the effects of Pasteurella multocida toxin (PMT), which activates osteoclasts and inhibits osteoblast activity, in C2C12 myoblasts expressing the mutant Alk2(R206H) receptor as model of FOP. In our study, we mainly used alkaline phosphatase (ALP) activity as marker to determine osteoblast differentiation. BMP-4 stimulated an increase in ALP activity in C2C12-Alk2wt and C2C12-Alk2(R206H) cells. By contrast, Activin A only induced ALP activity in C2C12-Alk2(R206H) cells. In both cases, PMT acted as a potent inhibitor of ALP activity. PMT-induced inhibition of ALP activity was paralleled by a constitutive activation of the heterotrimeric Gq protein. Expression of a permanently active Gαq blocked Activin A/Alk2(R206H)-dependent increase in ALP activity. Inactivation of Gq by specific inhibitor FR900359 blocked the PMT effect. Similarly, canonical second messengers and effectors of Gαq (e.g. ionophore A23187-induced increase in intracellular Ca2+ and activation of PKC by PMA (phorbol 12-myristate 13-acetate)) inhibited Alk2(R206H)-mediated induction of ALP activity. Notably, Activin A-induced increase in ALP activity in C2C12-Alk2(R206H) cells was also inhibited by stimulation of the α1A-adrenoceptor, which couples to Gαq, by phenylephrine. PMT did not alter tail phosphorylation of the major downstream effectors of the Alk2 receptor, Smad1/5/9; neither did the toxin affect nuclear translocation of the Smad-complex. However, PMT diminished BMP responsive element-induced gene expression. The data indicate that PMT potently inhibits the induction of osteoblast markers in a FOP model via activation of G proteins. Moreover, our findings indicate that activation of G protein-coupled receptors and of G protein signaling might be a rationale for pharmacological therapy of FOP.
Asunto(s)
Activinas/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Modelos Biológicos , Mioblastos/metabolismo , Miositis Osificante/patología , Osteoblastos/metabolismo , Transducción de Señal , Fosfatasa Alcalina/metabolismo , Animales , Proteína Morfogenética Ósea 4/metabolismo , Señalización del Calcio , Línea Celular , Ratones , Proteínas Smad/metabolismoRESUMEN
Salmonella enterica serotype Typhimurium (S. Typhimurium) is one of the most frequent causes of food-borne illness in humans and usually associated with acute self-limiting gastroenteritis. However, in immunocompromised patients, the pathogen can disseminate and lead to severe systemic diseases. S. Typhimurium are facultative intracellular bacteria. For uptake and intracellular life, Salmonella translocate numerous effector proteins into host cells using two type-III secretion systems (T3SS), which are encoded within Salmonella pathogenicity islands 1 (SPI-1) and 2 (SPI-2). While SPI-1 effectors mainly promote initial invasion, SPI-2 effectors control intracellular survival and proliferation. Here, we elucidate the mode of action of Salmonella SPI-2 effector SseI, which is involved in control of systemic dissemination of S. Typhimurium. SseI deamidates a specific glutamine residue of heterotrimeric G proteins of the Gαi family, resulting in persistent activation of the G protein. Gi activation inhibits cAMP production and stimulates PI3-kinase γ by Gαi-released Gßγ subunits, resulting in activation of survival pathways by phosphorylation of Akt and mTOR. Moreover, SseI-induced deamidation leads to non-polarized activation of Gαi and, thereby, to loss of directed migration of dendritic cells.
Asunto(s)
Proteínas Bacterianas/fisiología , Quimiotaxis , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Salmonella typhimurium , Sistemas de Secreción Tipo III/fisiología , Animales , Proteínas Bacterianas/genética , Supervivencia Celular/genética , Quimiotaxis/genética , Desaminación/genética , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Multimerización de Proteína/genética , Procesamiento Proteico-Postraduccional/genética , Células RAW 264.7 , Infecciones por Salmonella/metabolismo , Infecciones por Salmonella/patología , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismoRESUMEN
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)-, G12/13- 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 Gα12/13 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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Diferenciación Celular , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Sistema de Señalización de MAP Quinasas , Osteoblastos/metabolismo , Infecciones por Pasteurella/metabolismo , Pasteurella multocida/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Activación Transcripcional , Proteínas de Unión al GTP rho/metabolismo , Animales , Línea Celular , Ratones , Osteoblastos/patología , Osteólisis/metabolismo , Osteólisis/patología , Infecciones por Pasteurella/patología , Pasteurella multocida/patogenicidad , Ratas , Rinitis Atrófica/metabolismo , Rinitis Atrófica/patología , Cráneo/metabolismo , Cráneo/patología , Células del Estroma/metabolismo , Células del Estroma/patología , Factores de Virulencia/metabolismo , Proteína de Unión al GTP rhoARESUMEN
Pasteurella multocida is the causative agent of a number of epizootic and zoonotic diseases. Its major virulence factor associated with atrophic rhinitis in animals and dermonecrosis in bite wounds is P. multocida toxin (PMT). PMT stimulates signal transduction pathways downstream of heterotrimeric G proteins, leading to effects such as mitogenicity, blockade of apoptosis, or inhibition of osteoblast differentiation. On the basis of Gα(i2), it was demonstrated that the toxin deamidates an essential glutamine residue of the Gα(i2) subunit, leading to constitutive activation of the G protein. Here, we studied the specificity of PMT for its G-protein targets by mass spectrometric analyses and by utilizing a monoclonal antibody, which recognizes specifically G proteins deamidated by PMT. The studies revealed deamidation of 3 of 4 families of heterotrimeric G proteins (Gα(q/11), Gα(i1,2,3), and Gα(12/13) of mouse or human origin) by PMT but not by a catalytic inactive toxin mutant. With the use of G-protein fragments and chimeras of responsive or unresponsive G proteins, the structural basis for the discrimination of heterotrimeric G proteins was studied. Our results elucidate substrate specificity of PMT on the molecular level and provide evidence for the underlying structural reasons of substrate discrimination.