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Dorsomorphin and LDN-193189 inhibit BMP-mediated Smad, p38 and Akt signalling in C2C12 cells

https://doi.org/10.1016/j.biocel.2010.07.018Get rights and content

Abstract

Bone morphogenetic proteins (BMPs) are key regulators of cell fate decisions during embryogenesis and tissue homeostasis. BMPs signal through a coordinated assembly of two types of transmembrane serine/threonine kinase receptors to induce Smad1/5/8 plus non-Smad pathways, such as MAPK and Akt. The recent discovery of BMP receptor inhibitors opened new avenues to study specific BMP signalling and to delineate this effect from TGF-β and Activin signalling. Here we present comprehensive and quantitative analyses on both canonical and non-Smad mediated BMP signalling under Dorsomorphin (DM) and LDN-193189 (LDN) treatment conditions. We demonstrate for the first time, that both compounds affect not only the Smad but also the non-Smad signalling pathways induced by either BMP2, BMP6 or GDF5. The activation of p38, ERK1/2 and Akt in C2C12 cells was inhibited by DM and LDN. In addition “off-target” effects on all branches of BMP non-Smad signalling are presented. From this we conclude that the inhibition of BMP receptors by DM and more efficiently by LDN-193189 affects all known BMP induced signalling cascades.

Introduction

Bone morphogenetic proteins (BMPs) were originally described as bone morphogens, but in recent years it became evident that they also participate in a variety of processes during embryogenesis, tissue development and tissue repair. BMPs bind to two types of transmembrane Ser/Thr kinase receptors (type I and type II receptors), which upon transphosphorylation activate downstream signalling cascades, such as Smad1/5/8, MAPK and Akt/PKB. Malfunction of the pathways causes diseases ranging form bone disease, vascular diseases, organ dystrophies to cancer. For this reason, nature has selected for complex mechanisms to fine tune and regulate this central signalling network. Secreted antagonists control the accessibility of the ligands for the receptors. Co-receptors modulate the transmission of the extracellular signal into the cytosol while cytoplasmic and nuclear proteins bind and therefore regulate the activity of downstream signalling components (Sieber et al., 2009).

To understand the underlying signalling mechanisms there is a strong need for experimental approaches to manipulate BMP signalling. Classical approaches include the use of wildtype and mutant ligands, antagonists, soluble receptors or their ectodomains, neutralizing antibodies or genetic approaches using gene silencing or protein overexpression. Also specific protein aptamers have been successfully used to inhibit TGF-β signalling (Zhao and Hoffmann, 2006).Another strategy to specifically modulate BMP signalling is the use of small molecule inhibitors (Hong and Yu, 2009). The chemical compound Dorsomorphin (DM), formerly described as Compound C, was identified in a high throughput screen in Zebrafish to lead to a disordered formation of the dorsal-ventral axis in Zebrafish embryos (Yu et al., 2008a). This dorsalized axial pattern reflected the phenotype of BMP-pathway mutants and therefore strongly indicated that Dorsomorphin abrogated proper BMP signalling (Mullins et al., 1996).

The family of BMP proteins is comprised of over 20 family members, which signal through a limited number of type I and type II receptors. Ligand binding to type I (either Alk1/ActRIb, Alk2/ActRIa, Alk3/BRIa and Alk6/BRIb) and to type II receptors (either BRII, ActRIIa or ActRIIb) leads to an intracellular activation of the Smad signalling cascade as well as to an activation of several non-Smad pathways. For BMP2 it has been shown that the mode of receptor oligomerisation is crucial for the initiation of Smad versus non-Smad signalling. Upon ligand binding to the preformed complex composed of both type I and type II receptor, the constitutive active kinase domain of the type II receptor phosphorylates and thereby activates the GS-Box of the type I receptor (Nohe et al., 2002). Activated type I receptor is able to phosphorylate regulatory Smads (Smad1/5/8), which then form a ternary complex with the common mediator Smad (Smad4) and are selectively retained in the nucleus to regulate BMP target gene transcription (Hill, 2009, Sieber et al., 2009).

Although the Smad pathway has been studied extensively much less is known about transcriptional regulation via non-Smad signalling cascades. BMPs were shown to activate various members of different MAPK pathways, such as p38, ERK1/2 and SAPK/JNK (Gallea et al., 2001, Guicheux et al., 2003). Activation of p38 occurs via TAK1, TAB1 and XIAP, which are all associated to the BMP type I receptor (Yamaguchi et al., 1999). Activation of several MAP kinases results in subsequent phosphorylation and thus in activation of their downstream targets, like ATF2, the c-AMP response element binding protein (CREB), c-Jun or c-Fos and in transcriptional regulation of BMP target genes such as osteopontin, ALP or collagen I (Lai and Cheng, 2002, Barneda-Zahonero et al., 2009). Another important non-Smad signalling pathway includes activation of Akt/PKB, one of the key players regulating e.g. cell survival, proliferation, nutrition metabolism and migration (Manning and Cantley, 2007). This pathway was also shown to be activated in C2C12 cells upon BMP2 stimulation (Gamell et al., 2008).

It was shown previously that Dorsomorphin blocks BMP induced Smad1/5/8 phosphorylation in a dose dependent manner, while having no effect on TGF-β or Activin induced Smad2/3 activation as well as on BMP induced p38 activation (Yu et al., 2008a). The heterocyclic core structure of Dorsomorphin binds with different affinities the ATP binding site in the kinase domain of the type I receptors (Alk1, Alk2, Alk3 and Alk6) and thus inhibits their kinase activity (Yu et al., 2008a, Wrighton et al., 2009). Since Dorsomorphin is known to generally inhibit AMP-activated kinase (AMPK) as well as the receptor tyrosine kinases for PDGF and VEGF (Yu et al., 2008b, Hao et al., 2010), treatment of mammalian cells with this inhibitor shows various “off-target” effects. This disadvantage could be partly overcome by the Dorsomorphin derivative LDN-193189 (LDN) that exhibits a much higher specificity for BMP receptors and can be used at lower concentrations (Yu et al., 2008b).

The murine mesenchymal precursor cell line C2C12 is a common model to study BMP signalling, undergoing rapid osteogenic differentiation under BMP stimulation. In this study, we sought a more detailed understanding of the small molecule inhibitors DM and LDN in these cells. We demonstrate for the first time, that Dorsomorphin and LDN-193189 inhibit both BMP induced Smad1/5/8 phosphorylation and BMP-mediated induction of the p38 MAPK, Erk1/2 and Akt pathway in C2C12 cells.

Section snippets

Cell culture and reagents

C2C12 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Biochrom AG, Berlin, Germany) supplemented with 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany), 2 mM glutamine and penicillin (100 units/ml)/streptomycin (10 μg/ml) (PAA). For experiments cells were seeded into 12-well plates in a density of 7.5 × 105 cells/well. The following day, cells were starved in DMEM supplemented with 0% FCS, 2 mM glutamine and penicillin (100 units/ml)/streptomycin (10 μg/ml) prior to inhibitor and

Activation of Smad1/5/8, p38 and Akt by different BMP family members is inhibited by Dorsomorphin and LDN-193189 in a dose dependent manner

The discovery of pharmacological tools to selectively target the BMP signalling pathway has been very useful in discriminating TGF-β and BMP signalling inputs in physiological processes. LDN-193189 was the result of a structure-activity relationship study of Dorsomorphin (Cuny et al., 2008), which was discovered previously as an inhibitor of BMP induced Smad1/5/8 phosphorylation (Yu et al., 2008a). It has been reported, that in pulmonary arterial smooth muscle cells (PASMCs) both DM and LDN

Discussion

The molecular mechanism by which BMPs, in particular BMP2, induces the Smad pathway is well understood and very distinct from the mechanism, by which the same ligand induces non-Smad signalling pathways, such as MAPK and Akt (Nohe et al., 2002, Hartung et al., 2006, Sieber et al., 2009). Small molecule inhibitors targeting the receptors are therefore extremely valuable tools to dissect these complex signalling structures. For their use in therapeutic interventions it is of special importance to

Acknowledgements

We thank members of the Knaus lab for vivid discussions and support and Matheusz Kolanczyk for helpful advice. BMP2 was a generous gift from Walter Sebald (Würzburg), BMP6 from Slobodan Vukicevic (Zagreb) and GDF5 (BioPharm, Heidelberg). This work was supported by the Deutsche Forschungsgemeinschaft (research grant SFB760 to PK and a BSRT fellowship to JK).

References (25)

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