Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and β-catenin signaling

Yuka Bannai,Melinda J Faulkner,Mengfei Ho,Leila R Aminova,Brenda A Wilson

doi:10.3389/fcimb.2012.00080

Yuka Bannai, Melinda J Faulkner + Show 3 more

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https://doi.org/10.3389/fcimb.2012.00080

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Abstract

The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric Gq, Gi, and G12/13 proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of β-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and β-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G12/13 signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/β-catenin and Notch1 signaling, and in particular the role of Gq and G12/13 in mediating PMT effects on Rho/ROCK and Notch1 signaling.

Highlights

We previously demonstrated that Pasteurella multocida (PMT) potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of β-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development
Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/Rho-associated protein kinase (ROCK) signaling in mediating toxin effects on Wnt/β-catenin and Notch1 signaling, and in particular the role of Gq and G12/13 in mediating PMT effects on Rho/ROCK and Notch1 signaling
The 3T3-L1 cell line is a well-established in vitro model of adipocyte differentiation, which is characterized by expression of adipocyte-specific markers, such as PPARγ and C/EBPα (Rosen and Spiegelman, 2000; Otto and Lane, 2005), and downregulation of pre-adipocyte-specific markers, such as Pref1/Dlk1 (Wang et al, 2010)

Summary

Introduction

Members of the dermonecrotic toxin family of large G-proteinmodifying bacterial protein toxins are considered the causative agents or are associated with the major symptoms of a number of epizootic and zoonotic diseases (Roop et al, 1987; De Rycke et al, 1990; Woolfrey and Moody, 1991; Blanco et al, 1992; Foged, 1992; Horiguchi, 2001; Brockmeier et al, 2002; Arashima and Kumasaka, 2005; Diavatopoulos et al, 2005; Knust and Schmidt, 2010; Wilson and Ho, 2011). CNF1, CNF2, and CNF3 deamidate a specific Gln residue at the active site of the Rho GTPases, RhoA (Gln63), Rac (Gln61), and Cdc (Gln61) (Hoffmann and Schmidt, 2004; Knust and Schmidt, 2010), while CNFY acts only on RhoA (Gln63) (Hoffmann et al., 2004). The dermonecrotic toxin from Pasteurella multocida (PMT) activates small Rho GTPases (Ohnishi et al, 1998; Orth et al, 2005), but only indirectly through deamidation of analogous active site Gln residues in the α subunits of its target heterotrimeric G proteins, Gi (Gln205), Gq (Gln209), or G12/13 (Gln229) (Orth et al, 2005, 2009; Kamitani et al, 2011; Wilson and Ho, 2011). All of the dermonecrotic toxins are deamidases and DNT and the CNFs share strong sequence similarity in their catalytic domains, PMT does not have any sequence or structural similarity with DNT or the CNFs (Wilson and Ho, 2010, 2011)

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