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

Transforming growth factor-␤ (TGF-␤) elicits cellular effects by activating specific Smad proteins that control the transcription of target genes. Whereas there is growing evidence that there are TGF-␤ type I receptor-initiated intracellular pathways that are distinct from the pivotal Smad pathway, their physiological importance in TGF-␤ signaling is not well understood. Therefore, we generated TGF-␤ type I receptors (also termed ALK5s) with mutations in the L45 loop of the kinase domain, termed ALK5(D266A) and ALK5(3A). These mutants showed retained kinase activity but were unable to activate Smads. Characterization of their signaling properties revealed that the two L45 loop mutants did not mediate Smad-dependent transcriptional responses, TGF-␤-induced growth inhibition, and fibronectin and plasminogen activator-1 production in R4-2 mink lung epithelial cells lacking functional ALK5 protein. Mutation in the L45 loop region did not affect the binding of inhibitory Smads but did abrogate the weak binding of X-linked inhibitor of apoptosis protein and Disabled-2 to ALK5. This suggests that the L45 loop in the kinase domain is important for docking of other binding proteins. Interestingly, JNK MAP kinase activity was found to be activated by the ALK5(3A) mutant in various cell types. In addition, TGF-␤-induced inhibition of cyclin D1 expression and stimulation of PMEPA1 (androgenregulated prostatic mRNA) expression were found to occur, albeit weakly, in an Smad-independent manner in normal murine mammary gland cells. However, the TGF-␤-induced epithelial to mesenchymal transdifferentiation was found to require an intact L45 loop and is likely to be dependent on the Smad pathways.

ABSTRACT:Introduction: TGF-␤ is a multifunctional regulator of chondrocyte proliferation, differentiation, and extracellular matrix production. Dysregulation of TGF-␤ action has been implicated in cartilage diseases such as osteoarthritis. TGF-␤ signaling is transduced through a pair of transmembrane serine/threonine kinases, known as the type I (ALK5) and type II receptors. However, recent studies on endothelial cells have identified ALK1 as a second type I TGF-␤ receptor and have shown that ALK1 and ALK5 have opposing functions in these cells. Here we examined ALK1 expression and its regulation of TGF-␤ signaling and responses in human chondrocytes. Materials and Methods: ALK1 expression in human chondrocytes was examined by RT-PCR and Western blot. The ability of ALK1 to form complexes with other TGF-␤ receptors was determined by affinity labeling/ immunoprecipitation and by immunoprecipitation followed by Western blot. The effect of ALK1 on TGF-␤1-induced signaling and responses was determined by varying ALK1 expression levels and measuring transcriptional activity using promoter/luciferase assays, Smad1/5 and Smad3 phosphorylation, and expression of type II collagen, PAI-1, and fibronectin. Results: Our results indicate that ALK1 is expressed in human chondrocytes and that it is a component of the TGF-␤ receptor system, associating with ALK5, type II TGF-␤ receptor, endoglin, and betaglycan. Furthermore, we show that both ALK1 and ALK5 are needed for TGF-␤-induced phosphorylation of intracellular mediators Smad1/5, whereas only ALK5 is essential for TGF-␤1-induced phosphorylation of Smad3. In addition, our results show that ALK1 inhibits, whereas ALK5 potentiates, TGF-␤-induced Smad3-driven transcriptional activity and the expression of PAI-1, fibronectin, and type II collagen in chondrocytes. Conclusions: Our results suggest that ALK1 and ALK5 display opposing functions in human chondrocytes, implicating an essential role for ALK1 in the regulation of TGF-␤ signaling and function in these cells.

Progressive kidney fibrosis precedes end-stage renal failure in up to a third of patients with diabetes mellitus. Elevated intra-renal transforming growth factor-beta (TGF-beta) is thought to underlie disease progression by promoting deposition of extracellular matrix and epithelial-mesenchymal transition. GW788388 is a new TGF-beta type I receptor inhibitor with a much improved pharmacokinetic profile compared with SB431542. We studied its effect in vitro and found that it inhibited both the TGF-beta type I and type II receptor kinase activities, but not that of the related bone morphogenic protein type II receptor. Further, it blocked TGF-beta-induced Smad activation and target gene expression, while decreasing epithelial-mesenchymal transitions and fibrogenesis. Using db/db mice, which develop diabetic nephropathy, we found that GW788388 given orally for 5 weeks significantly reduced renal fibrosis and decreased the mRNA levels of key mediators of extracellular matrix deposition in kidneys. Our study shows that GW788388 is a potent and selective inhibitor of TGF-beta signalling in vitro and renal fibrosis in vivo.

Members of the transforming growth factor β (TGF-β) family have been genetically linked to vascular formation during embryogenesis. However, contradictory studies about the role of TGF-β and other family members with reported vascular functions, such as bone morphogenetic protein (BMP) 9, in physiological and pathological angiogenesis make the need for mechanistic studies apparent. We demonstrate, by genetic and pharmacological means, that the TGF-β and BMP9 receptor activin receptor-like kinase (ALK) 1 represents a new therapeutic target for tumor angiogenesis. Diminution of ALK1 gene dosage or systemic treatment with the ALK1-Fc fusion protein RAP-041 retarded tumor growth and progression by inhibition of angiogenesis in a transgenic mouse model of multistep tumorigenesis. Furthermore, RAP-041 significantly impaired the in vitro and in vivo angiogenic response toward vascular endothelial growth factor A and basic fibroblast growth factor. In seeking the mechanism for the observed effects, we uncovered an unexpected signaling synergy between TGF-β and BMP9, through which the combined action of the two factors augmented the endothelial cell response to angiogenic stimuli. We delineate a decisive role for signaling by TGF-β family members in tumor angiogenesis and offer mechanistic insight for the forthcoming clinical development of drugs blocking ALK1 in oncology.

Background: ALK1 is a TGF-␤ receptor essential for angiogenesis. An anti-ALK1 antibody is being tested in clinical trials as an inhibitor for tumor angiogenesis. Results: Anti-hALK1 antibody selectively recognizes ALK1 and interferes with ALK1 ligand binding, signaling, and endothelial sprouting. Conclusion: Anti-hALK1 antibody is a function-blocking antibody that prevents ALK1 signaling. Significance: This study shows mechanistically how anti-hALK1 antibody exerts its effect on angiogenesis.

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Endoglin (ENG) is a coreceptor of the transforming growth factor-β (TGFβ) family signaling complex, which is highly expressed on endothelial cells and plays a key role in angiogenesis. Its extracellular domain can be cleaved and released into the circulation as soluble ENG (sENG). High circulating levels of sENG contribute to the pathogenesis of preeclampsia (PE). Circulating bone morphogenetic protein 9 (BMP9), a vascular quiescence and endothelial-protective factor, binds sENG with high affinity, but how sENG participates in BMP9 signaling complexes is not fully resolved. sENG was thought to be a ligand trap for BMP9, preventing type II receptor binding and BMP9 signaling. Here we show that, despite cell-surface ENG being a dimer linked by disulfide bonds, sENG purified from human placenta and plasma from PE patients is primarily in a monomeric form. Incubating monomeric sENG with the circulating form of BMP9 (prodomain-bound form) in solution leads to the release of the prodomain and formation of a sENG:BMP9 complex. Furthermore, we demonstrate that binding of sENG to BMP9 does not inhibit BMP9 signaling. Indeed, the sENG:BMP9 complex signals with comparable potency and specificity to BMP9 on human primary endothelial cells. The full signaling activity of the sENG:BMP9 complex required transmembrane ENG. This study confirms that rather than being an inhibitory ligand trap, increased circulating sENG might preferentially direct BMP9 signaling via cell-surface ENG at the endothelium. This is important for understanding the role of sENG in the pathobiology of PE and other cardiovascular diseases.