Abstract 1539: ERK Signalling is Necessary for BMP-4 Dependent Capillary Sprouting and is Controlled by a Negative Feedback Loop Involving Smad6
Bone Morphogenetic Protein-4 (BMP-4) has a wide range of biological activities on vascular development and differentiation. Classically, BMPs mediate signals through the Smad pathway. During mouse development, lack of BMP-4 results in an earlier vascular phenotype than inactivation of Smad1 or 5 transcription factors. These observations suggest that BMPs also signal through other pathways in vascular cells.
Methods HUVECs were treated with BMP-4 and/or MEK inhibitors PD98059 and U0126. Smad6 was overxpressed using pcDNA3-Flag(N)-mSmad6. Constitutively active MEK1 (caMEK1) was overexpressed using a recombinant adenovirus. Cell lysates were analyzed by Western blotting. To investigate functional effect of BMP-4 treatment we generated HUVEC spheroids. In-gel angiogenesis was quantified by measuring the cumulative length of all capillary-like sprouts.
Results Incubation of HUVECs with BMP-4 results in a dose- and time-dependent activation of ERK1/2 in addition to the activation of the classical Smad pathway. Smad activation occurs slightly earlier than ERK phosphorylation. Phospho ERK increases 5.6 fold after stimulation with BMP-4 (50 ng/ml) for 20 min compared to untreated cells. This effect but not phosphorylation of Smad1/5 is completely blocked by pre-incubation of cells with MEK inhibitors. Quantitative analysis of capillary sprouting demonstrate a reduction of sprout length of 74% (p<0.001) in the presence of MEK inhibitors, indicating that ERK signals are necessary for BMP signal transduction. Overexpression of caMEK1 results in Smad1/5 phosphorylation confirming a positive feedback loop between Smad and ERK signalling cascades. Conversely, we found that overexpression of the inhibitory Smad6 blocked ERK phosphorylation in response to BMP-4.
Conclusions We demonstrate that ERK1/2 functions as an alternative pathway in BMP-4 signalling in HUVECs. Capillary sprouting induced by BMP-4 is dependent on ERK phosphorylation. Our data indicate that the ERK pathway is essential for efficient transduction of BMP signals and serves as a positive feedback mechanism. On the other hand, excessive stimulation of Smads including Smad6 attenuates ERK activation and thus results in a negative feedback loop to fine-tune BMP signalling in HUVECs.