TGFβ Signal Transduction in Endothelial Cells

Sammanfattning: Transforming growth factor β (TGFβ) is a multifunctional cytokine that is involved in many biological effects, such as proliferation, migration, differentiation and cell survival. TGFβ regulates cellular responses by binding to a heteromeric complex of type I and type II serine/threonine kinase receptors. The type I receptor, termed activin receptor-like kinase (ALK), acts downstream of the type II receptor and propagates the signal to the nucleus by phosphorylating receptor regulated Smads (R-Smads). The activated R-Smads can associate with the common partner Smad, Smad4, and this complex translocates to the nucleus where it participates in transcriptional regulation of target genes. TGFβ plays an important role in vascular morphogenesis. The aim of this study was to obtain more insight into the mechanisms by which TGFβ can act as an inhibitor or stimulator of angiogenesis Our findings show that in endothelial cells (ECs), TGFβ can activate two distinct type I receptor/Smad signalling pathways with opposite cellular responses. In most cell types, TGFβ signals via the TGFβ type I receptor, ALK5. However, ECs express a predominant endothelial type I receptor, named ALK1. Whereas the TGFβ/ALK1 signalling leads to activation, the TGFβ/ALK5 pathway results in an inhibition of the activation state. This suggests that TGFβ regulates the activation state of the endothelium via a fine balance between these two pathways. We identified genes that are specifically induced by TGFβ mediated ALK1 or ALK5 activation. Id1 was found to be the target gene of the ALK1/Smad1/5 pathway while induction of plasminogen activator inhibitor-1 was activated only by ALK5/Smad2 pathway. Furthermore, ALK1 activated ECs are highly invasive but this property is lost if Id1 expression is specifically knocked-down. ECs invasiveness is highly dependent on αv integrin binding to its extracellular matrix (ECM) protein partner and the invasion requires proteolytic cleavage of the ECM by metalloproteases (MMPs). Hence, TGFβ/ALK1/Id1 pathway may promote invasion by modulating the expression or activity of integrins and MMPs that are well known components of the ECM. Timing and duration of TGFβ signalling are important specificity determinants for its effect on cellular behaviour. After binding to ALK1, TGFβ induces a transient phosphorylation of Smad1/5 but a stable phosphorylation of Smad2 via ALK5. Our studies indicate that Smad7 is potently induced by ALK1 signalling and may recruit a PP1α/TIMAP phosphatase complex to ALK1 to dephosphorylate the receptor and thereby turning off phosphorylation resulting in a temporal activation of TGFβ/ALK1-induced Smad1/5 pathway. This mechanism enables an efficient and tightly temporally controlled activation resulting in the dominance of ALK5 upon prolonged exposure to TGFβ. Bone morphogenetic protein (BMP) is a member of the TGFβ superfamily and signals through Smad1/5. The BMP/Smad1/5 pathway was found to potently activate the endothelium. Id1 was identified as an important BMP target gene in ECs and was sufficient and necessary for BMP-induced EC migration. These studies not only provide new insights into possible molecular mechanisms that underlie activation and quiescence of ECs during physiological angiogenesis but may also explain the vascular phenotypes observed in mice and humans with perturbed TGFβ signalling pathways.