ALK5- and TGFBR2-independent role of ALK1 in the pathogenesis of hereditary hemorrhagic telangiectasia type 2.
ABSTRACT ALK1 belongs to the type I receptor family for transforming growth factor-beta family ligands. Heterozygous ALK1 mutations cause hereditary hemorrhagic telangiectasia type 2 (HHT2), a multisystemic vascular disorder. Based largely on in vitro studies, TGF-beta1 has been considered as the most likely ALK1 ligand related to HHT, yet the identity of the physiologic ALK1 ligand remains controversial. In cultured endothelial cells, ALK1 and another TGF-beta type I receptor, ALK5, regulate angiogenesis by controlling TGF-beta signal transduction, and ALK5 is required for ALK1 signaling. However, the extent to which such interactions between these 2 receptors play a role in pathogenesis of HHT is unknown. We directly addressed these issues in vivo by comparing the phenotypes of mice in which the Alk1, Alk5, or Tgfbr2 gene was conditionally deleted in restricted vascular endothelia using a novel endothelial Cre transgenic line. Alk1-conditional deletion resulted in severe vascular malformations mimicking all pathologic features of HHT. Yet Alk5- or Tgfbr2-conditional deletion in mice, or Alk5 inhibition in zebrafish, did not affect vessel morphogenesis. These data indicate that neither ALK5 nor TGFBR2 is required for ALK1 signaling pertinent to the pathogenesis of HHT and suggest that HHT might not be a TGF-beta subfamily disease.
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ABSTRACT: Germ line mutations in one of two distinct genes, endoglin or ALK-1, cause hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant disorder of localized angiodysplasia. Both genes encode endothelial cell receptors for the transforming growth factor beta (TGF-beta) ligand superfamily. Endoglin has homology to the type III receptor, betaglycan, although its exact role in TGF-beta signaling is unclear. Activin receptor-like kinase 1 (ALK-1) has homology to the type I receptor family, but its ligand and corresponding type II receptor are unknown. In order to identify the ligand and type II receptor for ALK-1 and to investigate the role of endoglin in ALK-1 signaling, we devised a chimeric receptor signaling assay by exchanging the kinase domain of ALK-1 with either the TGF-beta type I receptor or the activin type IB receptor, both of which can activate an inducible PAI-1 promoter. We show that TGF-beta1 and TGF-beta3, as well as a third unknown ligand present in serum, can activate chimeric ALK-1. HHT-associated missense mutations in the ALK-1 extracellular domain abrogate signaling. The ALK-1/ligand interaction is mediated by the type II TGF-beta receptor for TGF-beta and most likely through the activin type II or type IIB receptors for the serum ligand. Endoglin is a bifunctional receptor partner since it can bind to ALK-1 as well as to type I TGF-beta receptor. These data suggest that HHT pathogenesis involves disruption of a complex network of positive and negative angiogenic factors, involving TGF-beta, a new unknown ligand, and their corresponding receptors.Journal of Biological Chemistry 05/1999; 274(15):9984-92. · 4.65 Impact Factor
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ABSTRACT: ALK-1 (activin receptor-like kinase-1), a type I receptor of the transforming growth factor (TGF)-beta superfamily, is the gene mutated in hereditary hemorrhagic telangiectasia type 2 (HHT2) while endoglin is mutated in HHT1. Using a novel polyclonal antibody to ALK-1, we measured ALK-1 expression on human umbilical vein endothelial cells (HUVEC) of newborns from HHT families whose affected members had normal endoglin levels. ALK-1 levels were specifically reduced in three HUVEC with ALK-1 missense mutant codons, and normal in two newborns not carrying the missense mutations present in the clinically affected relatives. Levels were also normal in a HUVEC with deletion of S232 in the ATP binding site of ALK-1. Thus HHT2 appears to be associated with a loss of function of the mutant allele due to a reduction in either protein level or activity. We also report three new ALK-1 missense mutations leading to G48E/A49P, C344Y and E407D substitutions. In COS-1 transfected cells, ALK-1 was found in the TGF-beta1 and -beta3 receptor complexes in association with endoglin and TbetaRII, but not in activin receptor complexes containing endoglin. In HUVEC, ALK-1 was not detectable in the TGF-beta1 or -beta3 receptor complexes. However, in the absence of ligand, ALK-1 and endoglin interactions were observed by immunoprecipitation/western blot in HUVEC from normal as well as HHT1 and HHT2 patients. Our data suggest a transient association between these two proteins of the TGF-beta superfamily, both required at a critical level to ensure vessel wall integrity.Human Molecular Genetics 06/2000; 9(8):1227-37. · 7.69 Impact Factor
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ABSTRACT: Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth, LacZ activity was detected in most endothelial cells within the embryo and of extra-embryonic tissues such as yolk sac and chorioallantoic placenta. Ectopic expression of Cre was observed in approximately 12-20% of the adult erythroid, myeloid and lymphoid cells and in subregions of the adult brain. These results show that the tie-1-Cre transgenic strain can efficiently direct deletion of floxed genes in endothelial cells in vivo.Journal of Cell Science 03/2001; 114(Pt 4):671-6. · 5.88 Impact Factor