Marie-José Goumans

Balancing the activation state of the endothelium via two distinct TGF‐β type I receptors

The generation of mice lacking specific components of the transforming growth factor‐β (TGF‐β) signal tranduction pathway shows that TGF‐β is a key player in the development and physiology of the cardiovascular system. Both pro‐ and anti‐angiogenic properties have been ascribed to TGF‐β, for which the molecular mechanisms are unclear. Here we report that TGF‐β can activate two distinct type I receptor/Smad signalling pathways with opposite effects. TGF‐β induces phosphorylation of Smad1/5 and Smad2 in endothelial cells and these effects can be blocked upon selective inhibition of ALK1 or ALK5 expression, respectively. Whereas the TGF‐β/ALK5 pathway leads to inhibition of cell migration and proliferation, the TGF‐β/ALK1 pathway induces endothelial cell migration and proliferation. We identified genes that are induced specifically by TGF‐β‐mediated ALK1 or ALK5 activation. Id1 was found to mediate the TGF‐β/ALK1‐induced (and Smad‐dependent) migration, while induction of plasminogen activator inhibitor‐1 by activated ALK5 may contribute to the TGF‐β‐induced maturation of blood vessels. Our results suggest that TGF‐β regulates the activation state of the endothelium via a fine balance between ALK5 and ALK1 signalling.

Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFβ/ALK5 signaling

Transforming growth factor-beta (TGFbeta) regulates the activation state of the endothelium via two opposing type I receptor/Smad pathways. Activin receptor-like kinase-1 (ALK1) induces Smad1/5 phosphorylation, leading to an increase in endothelial cell proliferation and migration, while ALK5 promotes Smad2/3 activation and inhibits both processes. Here, we report that ALK5 is important for TGFbeta/ALK1 signaling; endothelial cells lacking ALK5 are deficient in TGFbeta/ALK1-induced responses. More specifically, we show that ALK5 mediates a TGFbeta-dependent recruitment of ALK1 into a TGFbeta receptor complex and that the ALK5 kinase activity is required for optimal ALK1 activation. TGFbeta type II receptor is also required for ALK1 activation by TGFbeta. Interestingly, ALK1 not only induces a biological response opposite to that of ALK5 but also directly antagonizes ALK5/Smad signaling.

Endoglin promotes endothelial cell proliferation and TGF-β/ALK1 signal transduction

Endoglin is a transmembrane accessory receptor for transforming growth factor‐β (TGF‐β) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF‐β signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor‐like kinase 1 (ALK1), an endothelial specific TGF‐β type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF‐β/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF‐β/ALK1 signalling, which indirectly inhibits TGF‐β/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF‐β/ALK1 signalling is reduced and TGF‐β/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF‐β‐induced growth arrest by indirectly reducing TGF‐β/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.

Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice.

Deletion of the transforming growth factor β1 (TGF‐β1) gene in mice has previously suggested that it regulates both hematopoiesis and angiogenesis. To define the function of TGF‐β more precisely, we inactivated the TGF‐β type I receptor (TβRI) gene by gene targeting. Mice lacking TβRI die at midgestation, exhibiting severe defects in vascular development of the yolk sac and placenta, and an absence of circulating red blood cells. However, despite obvious anemia in the TβRI−/− yolk sacs, clonogenic assays on yolk sac‐derived hematopoietic precursors in vitro revealed that TβRI−/− mice exhibit normal hematopoietic potential compared with wild‐type and heterozygous siblings. Endothelial cells derived from TβRI‐deficient embryos show enhanced cell proliferation, improper migratory behavior and impaired fibronectin production in vitro, defects that are associated with the vascular defects seen in vivo. We thus demonstrate here that, while TβRI is crucial for the function of TGF‐β during vascular development and can not be compensated for by the activin receptor‐like kinase‐1 (ALK‐1), functional hematopoiesis and development of hematopoietic progenitors is not dependent on TGF‐β signaling via TβRI.

Regulation of cell proliferation by Smad proteins

Transforming growth factor‐β (TGF‐β) family members which include TGF‐βs, activins, and bone morphogenetic proteins (BMPs) regulate a broad spectrum of biological responses on a large variety of cell types. TGF‐β family members initiate their cellular responses by binding to distinct receptors with intrinsic serine/threonine kinase activity and activation of specific downstream intracellular effectors termed Smad proteins. Smads relay the signal from the cell membrane to the nucleus, where they affect the transcription of target genes. Smad activation, subcellular distribution, and stability have been found to be intricately regulated and a broad array of transcription factors have been identified as Smad partners. Important activities of TGF‐β are its potent anti‐mitogenic and pro‐apoptotic effects that, at least in part, are mediated via Smad proteins. Escape from TGF‐β/Smad‐induced growth inhibition and apoptosis is frequently observed in tumors. Certain Smads have been found to be mutated in specific types of cancer and gene ablation of particular Smads in mice has revealed increased rate of tumorigenesis. In late stage tumors, TGF‐β has been shown to function as a tumor promoter. TGF‐β can stimulate the de‐differentiation of epithelial cells to malignant invasive and metastatic fibroblastic cells. Interestingly, TGF‐β may mediate these effects directly on tumor cells via subverted Smad‐dependent and/or Smad‐independent pathways. J. Cell. Physiol. 191: 1–16, 2002.

Stimulation of id1 expression by bone morphogenetic protein is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells.

Background—Bone morphogenetic proteins (BMPs) are multifunctional proteins that regulate the proliferation, differentiation, and migration of a large variety of cell types. Like other members of the transforming growth factor-&bgr; family, BMPs elicit their cellular effects through activating specific combinations of type I and type II serine/threonine kinase receptors and their downstream effector proteins, which are termed Smads. In the present study, we investigated BMP receptor/Smad expression and signaling in endothelial cells (ECs) and examined the effects of BMP on EC behavior. Methods and Results—Immunohistochemical analysis of tissue sections of human colon and mouse heart and aorta showed that BMP receptors are expressed in ECs in vivo. Bovine aortic ECs and mouse embryonic ECs were found to express BMP receptors and their Smads. BMP receptor activation induced the phosphorylation of specific Smad proteins and promoted EC migration and tube formation. Id1 was identified as a BMP/Smad target in ECs. Ectopic expression of Id1 mimicked BMP-induced effects. Importantly, specific interference with Id1 expression blocked BMP-induced EC migration. Conclusions—The BMP/Smad pathway can potently activate the endothelium. Id1 expression is strongly induced by BMP in ECs. Ectopic expression of Id1 induces EC migration and tube formation. Moreover, Id1 played a critical role in mediating BMP-induced EC migration.

Controlling the angiogenic switch: a balance between two distinct TGF-b receptor signaling pathways.

Biochemical studies in endothelial cells (ECs) and genetic studies in mice and humans have yielded major insights into the role of transforming growth factor beta (TGF-beta) and its downstream Smad effectors in embryonic vascular morphogenesis and in the establishment and maintenance of vessel wall integrity. These studies showed that TGF-beta signaling is of critical importance for normal vascular development and physiology. They also indicated the involvement of two distinct TGF-beta signaling cascades within ECs, namely the activin receptor-like kinase 5 (ALK5)-Smad2/3 pathway and the ALK1-Smad1/5 pathway. Aberrant TGF-beta signaling forms the basis for several vascular disorders such as hereditary hemorrhagic telengiectasia and primary pulmonary hypertension as well as neovascularization during tumorigenesis. This review describes the role of TGF-beta in angiogenesis and some of the controversial issues concerning TGF-beta signaling through ALK1 and ALK5 in ECs.

Transforming growth factor β signal transduction

Transforming growth factor beta1 (TGF‐β1) is the prototypic member of a large family of structurally related pleiotropic‐secreted cytokines that play a pivotal role in the control of differentiation, proliferation, and state of activation of many different cell types including immune cells. TGF‐β family members have potent immunosuppressor activities in vitro and in vivo. These cytokines trigger their biological effects by inducing the formation of a heteromeric transmembrane serine/threonine kinase receptor complex. These receptors then initiate intracellular signaling through activation of Smad proteins, and specific Smads become phosphorylated and associate with other Smads. These heteromeric Smad complexes accumulate in the nucleus, where they modulate the expression of target genes. Recent data support the notion that Smads are important intracellular effectors of TGF‐β in immune cells. Here, we review recent advances in TGF‐β signal transduction in immune cells.

Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells

Notch and bone morphogenetic protein signaling pathways are important for cellular differentiation, and both have been implicated in vascular development. In many cases the two pathways act similarly, but antagonistic effects have also been reported. The underlying mechanisms and whether this is caused by an interplay between Notch and BMP signaling is unknown. Here we report that expression of the Notch target gene, Herp2, is synergistically induced upon activation of Notch and BMP receptor signaling pathways in endothelial cells. The synergy is mediated via RBP‐Jκ/CBF‐1 and GC‐rich palindromic sites in the Herp2 promoter, as well as via interactions between the Notch intracellular domain and Smad that are stabilized by p/CAF. Activated Notch and its downstream effector Herp2 were found to inhibit endothelial cell (EC) migration. In contrast, BMP via upregulation of Id1 expression has been reported to promote EC migration. Interestingly, Herp2 was found to antagonize BMP receptor/Id1‐induced migration by inhibiting Id1 expression. Our results support the notion that Herp2 functions as a critical switch downstream of Notch and BMP receptor signaling pathways in ECs.

Controlling cell fate by bone morphogenetic protein receptors

Bone morphogenetic proteins (BMPs) are multifunctional proteins that regulate the fate of different cell types, including mesenchymal and endothelial cells. BMPs inhibit myogenic differentiation, but promote the differentiation of mesenchymal cells into osteoblasts. Furthermore, endothelial migration and tube formation are stimulated by BMPs. Like other members of the transforming growth factor-beta (TGF-beta) superfamily, BMPs elicit their cellular effects via specific types I and II serine/threonine receptors. The activated BMP type I receptor phosphorylates specific receptor-regulated (R)-Smad proteins, which assemble into heteromeric complexes with common partner (Co)-Smad4. Heteromeric Smad complexes efficiently translocate into the nucleus, where they regulate the transcription of target genes. Inhibitors of differentiation (Id) are genes that are specifically induced by BMPs in tissues of different origin. Promoter analysis of Id1 indicates three distinct sequence elements that are sufficient and essential for efficient BMP-induced activation. Furthermore, recent studies reveal an important effector function for Id1 in various BMP-induced biological responses.