ERK signaling is a central regulator for BMP-4 dependent capillary sprouting.
OBJECTIVE
Bone Morphogenetic Protein-4 (BMP-4) and Extracellular-Signal Regulated Kinases (ERK) play crucial roles in vascular diseases. Here, we demonstrate that BMP-4 not only signals through the classical Smad cascade but also activates ERK phosphorylation as an alternative pathway in human umbilical vein endothelial cells (HUVEC) and that Smad and ERK pathways communicate through signal crosstalk.


METHODS
HUVECs were treated with BMP-4 and/or MEK inhibitors. Smad 6 and constitutively active (ca) MEK1 were overexpressed. Loss of function of Smad 4 and Smad 6 was achieved by specific siRNA transfection. Cell lysates were analyzed by western blotting for Smad and ERK phosphorylation. HUVEC spheroids were generated for angiogenesis quantification.


RESULTS
Treatment with BMP-4 results in a dose- and time-dependent activation of the MEK-ERK 1/2 pathway in addition to activation of the Smad pathway and is blocked by MEK inhibitors. Quantitative in-gel angiogenesis assays in the presence or absence of MEK inhibitors demonstrate that ERK signals are necessary for BMP-4 induced capillary sprouting. Furthermore sprouting is not blocked by inhibition of the Smad signaling pathway. Overexpression of the inhibitory Smad 6 inhibits ERK phosphorylation and ERK-induced capillary sprouting, whereas loss of function of Smad 4 has no effect.


CONCLUSIONS
We demonstrate that ERK1/2 functions as an alternative pathway in BMP-4 signaling in HUVECs. Capillary sprouting induced by BMP-4 is dependent on ERK phosphorylation. ERK is essential for efficient transduction of BMP signals and serves as a positive feedback mechanism. On the other hand, stimulation of Smad 6 inhibits ERK activation and thus results in a negative feedback loop to fine-tune BMP signaling in HUVECs.
Zhou
Qian
Q
Department of Cardiology, University of Freiburg, Freiburg, Germany.
Heinke
Jennifer
J
Vargas
Alberto
A
Winnik
Stephan
S
Krauss
Tobias
T
Bode
Christoph
C
Patterson
Cam
C
Moser
Martin
M
D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism*; D002196:Capillaries / Q000166:cytology / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000502:physiology*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D020239:Receptor Cross-Talk / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D051905:Smad6 Protein / Q000378:metabolism; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism
C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D034741:RNA, Small Interfering; C497802:SMAD6 protein, human; D051905:Smad6 Protein; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse