Figure 1. Endoglin biology in endothelial cells. (central part) Endoglin (Eng) is a component of a receptor complex in endothelial cells comprising the type I receptor dimers activin receptor-like kinase 5 (ALK5) or ALK1, and type II receptor dimers binding ligands, e.g., transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 9 (BMP9), of the TGF-β-superfamily. Interaction with these receptors either activates Smad1/5/8 or Smad2/3 signaling (phosphorylation) to regulate corresponding target genes, e.g., inhibitor of differentiation 1 (Id1) and plasminogen activator inhibitor-1 (PAI-1), and finally, cellular responses including proliferation and migration to balance the activation and resolution phase of angiogenesis. Localization of Eng in caveolin-1 (cav-1) positive lipid rafts mediates interaction of heat shock protein 90 (Hsp90) with endothelial NO-synthase (eNOS) regulating its function. (upper part) Beside the long variant of Eng (L-Eng), a C-terminally truncated, shorter splice variant (S-Eng) and a shedded form, mediated by matrix metalloproteinase (MMP)-14, comprising only the extracellular domain (sEng) has been identified. All three variants of Eng are involved individually in cellular (senescence) and pathological conditions (e.g., HHT-1, pre-eclampsia), respectively. The extracellular domain of Eng is N- and O-glycosylated and human Eng contains an integrin binding motif (RGD) motif. The L-Eng C-terminal domain is a substrate for TGF-β receptors leading to serine/threonine phosphorylation. Tyrosine phosphorylation is mediated by Src. L-Eng interacts with its cytoplasmic domain with several intracellular proteins regulating receptor trafficking, phosphorylation/activity of kinases, and focal adhesion amongst others. The functional difference of S-Eng compared to L-Eng most likely arises from the missing binding- and phosphorylation-sites in its C-terminus. Abbreviations used are: GIPC, GAIP C-terminus-interacting protein; HHT-1, hereditary hemorrhagic telangiectasia 1; Smad, small mothers against decapentaplegic; pERK1/2, phosphorylated extracellular signal-regulated kinase 1/2; PI3K/Akt, phosphatidylinositol 3-kinase; TβRI/II, transforming growth factor-β1 type I or II receptor; ZRP-1, zyxin-related protein-1.

Beside the “regular” Endoglin variant (L-Endoglin), a shorter splice variant of Endoglin (S-Endoglin) has been identified [15,16,17]. This splice variant has a shortened C-terminus, which is missing phosphorylation and protein interaction sites, resulting in functional differences compared to L-Endoglin (see below). Upon ligand binding, TβRII and TβRI activation results in endocytosis of the receptor complex followed by phosphorylation of substrate proteins by TβRI. In general, there are two different ways of internalization involving clathrin-coated pits (ccp, early endosomal antigen (EEA)-1⁺) or alternatively caveolin-coated pits (lipid rafts, caveolin-1⁺). It is assumed that localization to ccp leads to Smad activation, whereas routing to lipid rafts leads to degradation of receptors or activation of non-Smad signaling [18,19]. In endothelial cells (EC), Endoglin is localized to the membrane of caveolae, where it regulates the stability and interaction of eNOS and its allosteric regulator Hsp90 [20]. In addition, Endoglin was found in detergent-insoluble proteins (representing most likely lipid rafts) in placentas of pre-eclamptic women [21]. Activated Smads translocate to the nucleus in complex with the common Smad4, and in concert with positive and negative transcription factors regulate a wide spectrum of target genes [22]. In ECs, Endoglin modulates diverse functions, including migration and proliferation, by differentially regulating Smad2/3 signaling versus Smad1/5/8 signaling, which involves the interaction of Endoglin with the structural protein GIPC [13,23]. Moreover, the phosphorylation of the C-terminal part of Endoglin generates docking sites for intracellular interacting proteins, which also applies to subsets of T-cells (see below) [24,25]. However, the intracellular signal transfer modalities to regulate transcription or influence cellular functions by Endoglin (at least in EC) are not restricted to Smad-dependent mechanisms [26,27].

Endoglin expression is upregulated in activated ECs and this is facilitated by different transcription factors, including TGF-β1 regulated Smad3/Sp1/KLF6 and hypoxia regulated Hif1α [28,29]. Due to the high expression of Endoglin in activated ECs, the function and biology of Endoglin has been analyzed in detail in ECs and processes based on angiogenesis including carcinogenesis [30]. Especially, Endoglin has been exploited to target anti-cancer drugs to the tumor vasculature to treat cancer [31,32]. In contrast to EC, in tumor cells of different origins, in which Endoglin acts as a tumor suppressor, its expression is epigenetically silenced/switched off [33,34,35,36].

The area of EC research was enforced by the finding that the human disease hereditary hemorrhagic telangiectasia 1 (HHT-1) is based on genetic defects in the endoglin gene (Eng), leading to defect/non-functional or mis-targeted proteins causing haploinsufficiency in Endoglin [1,37,38,39,40]. Several Endoglin mutants have been shown to get stuck in the endoplasmic reticulum due to folding/glycosylation defects [41]. Those proteins are most likely ubiquitinated, a fact substantiated by the interaction with the E3 ubiquitin ligase tripartite motif-containing protein 21 (TRIM21) [42]. Likewise, to the multiple and sometimes life-threatening complications in HHT-1 patients, the loss of Endoglin causes embryonic lethality in homozygous Endoglin knockout mice, underpinning the important function of Endoglin during development [43]. More importantly, Endoglin knockout mouse models revealed that not only EC but also vessel-associated cells express Endoglin, and the communication of these cells with EC is the basis for defective angiogenesis [44]. In addition, the function of Endoglin is not limited to Endoglin expressing cells, since this receptor itself can directly be a part of the paracrine communication by the proteolytical release of the extracellular domain as soluble Endoglin (sEng) from the cell surface [45]. sEng is increased in pre-eclamptic woman and has been functionally linked to this disease [46]. Moreover, sEng and the membrane bound full-length Endoglin can be released by cells as a cargo of exosomes [4].