Endoglin (ENG) is a type I membrane glycoprotein located on cell surfaces and is part of the TGF beta receptor complex. It is also commonly referred to as CD105, END, FLJ41744, HHT1, ORW and ORW1. [5] It has a crucial role in angiogenesis, therefore, making it an important protein for tumor growth, survival and metastasis of cancer cells to other locations in the body.
The human endoglin gene is located on human chromosome 9 with location of the cytogenic band at 9q34.11. [6] [7] Endoglin glycoprotein is encoded by 39,757 bp and translates into 658 amino acids. [5]
The expression of the endoglin gene is usually low in resting endothelial cells. This, however, changes once neoangiogenesis begins and endothelial cells become active in places like tumor vessels, inflamed tissues, skin with psoriasis, vascular injury and during embryogenesis. [5] The expression of the vascular system begins at about 4 weeks and continues after that. [5] Other cells in which endoglin is expressed consist of monocytes, especially those transitioning into macrophages, low expression in normal smooth muscle cells, high expression vascular smooth muscle cells and in kidney and liver tissues undergoing fibrosis. [5] [8]
The glycoprotein consists of a homodimer of 180 kDA stabilized by intermolecular disulfide bonds. [9] It has a large extracellular domain of about 561 amino acids, a hydrophobic transmembrane domain and a short cytoplasmic tail domain composed of 45 amino acids. [9] The 260 amino acid region closest to the extracellular membrane is referred to as the ZP domain (or, more correctly, ZP module). [10] [11] The outermost extracellular region is termed as the orphan domain (or, more correctly, orphan region (OR)) and it is the part that binds ligands such as BMP-9. [12] [13]
There are two isoforms of endoglin created by alternative splicing: the long isoform (L-endoglin) and the short isoform (S-endoglin). [14] However, the L-isoform is expressed to a greater extent than the S-isoform. A soluble form of endoglin can be produced by the proteolytic cleaving action of metalloproteinase MMP-14 in the extracellular domain near the membrane. [5] It has been found on endothelial cells in all tissues, [9] activated macrophages, activated monocytes, lymphoblasts fibroblasts, and smooth muscle cells. Endoglin was first identified using monoclonal antibody (mAb) 44G4 but more mAbs against endoglin have been discovered, giving more ways to identify it in tissues. [15]
It is suggested that endoglin has 5 potential N-linked glycosylation sites in the N-terminal domain (of which N102 was experimentally observed in the crystal structure of the orphan region ( PDB: 5I04 )) and an O-glycan domain near the membrane domain that is rich in Serine and Threonine. [9] The cytoplasmic tail contains a PDZ-binding motif that allows it to bind to PDZ containing proteins and interact with them. [16] It contains an Arginine-Glycine-Aspartic Acid (RGD) tripeptide sequence that enables cellular adhesion, through the binding of integrins or other RGD binding receptors that are present in the extracellular matrix (ECM). [9] This RGD sequence on endoglin is the first RGD sequence identified on endothelial tissue. [9]
X-ray crystallographic structures of human endoglin ( PDB: 5I04, 5HZV ) and its complex with ligand BMP-9 ( PDB: 5HZW ) revealed that the orphan region of the protein (residues E26-S337) consists of two domains (OR1 and OR2, corresponding to residues E36-T46 + T200-C330 and residues S47-R199, respectively) with a new fold resulting from gene duplication and circular permutation. [13] The ZP module (residues P338-G581), whose ZP-N and ZP-C moieties (residues T349-L443 and N444-S576, respectively) are closely packed against each other, mediates the homodimerization of endoglin by forming an intermolecular disulfide bond that involves cysteine 516. [13] Together with a second intermolecular disulfide, involving cysteine 582, [16] this generates a molecular clamp that secures the ligand via interaction of two copies of OR1 with the knuckle regions of homodimeric BMP-9. [13] In addition to rationalizing a large number of HHT1 mutations, the crystal structure of endoglin shows that the epitope of anti-ENG monoclonal antibody TRC105 overlaps with the binding site for BMP-9. [13]
Endoglin has been shown to interact with high affinity to TGF beta receptor 3 [6] [17] and TGF beta receptor 1, [16] [18] and with lower affinity to TGF beta receptor 2. [6] It has high sequence similarity to another TGF beta binding protein, betaglycan, which was one of the first cues that indicated that endoglin is a TGF beta binding proteins. [19] However, it has been shown that TGF beta binds with high affinity to only a small amount of the available endoglin, which suggests that there is another factor regulating this binding. [19]
Endoglin itself doesn't bind the TGF beta ligands, but is present with the TGF beta receptors when the ligand is bound, indicating an important role for endoglin. [16] The full length endoglin will bind to the TGF beta receptor complex whether TGF beta is bound or not, but the truncated forms of endoglin have more specific binding. [16] The amino acid (aa) region 437–558 in the extracellular domain of endoglin will bind to TGF beta receptor II. TGF beta receptor I binds to the 437-588 aa region and to the aa region between 437 and the N-terminus. [16] Unlike TGF beta receptor I which can only bind the cytoplasmic tail when its kinase domain is inactive, TGF beta receptor II can bind endoglin with an inactive and active kinase domain. [16] The kinase is active when it is phosphorylated. Furthermore, TGF beta receptor I will dissociate from endoglin soon after it phosphorylates its cytoplasmic tail, leaving TGF beta receptor I inactive. [16] Endoglin is constituitively phosphorylated at the serine and threonine residues in the cytoplasmic domain. The high interaction between endoglin's cytoplasmic and extracellular tail with the TGF beta receptor complexes indicates an important role for endoglin in the modulation of the TGF beta responses, such as cellular localization and cellular migration. [16]
Endoglin can also mediate F-actin dynamics, focal adhesions, microtubular structures, endocytic vesicular transport through its interaction with zyxin, ZRP-1, beta-arrestin and Tctex2beta, LK1, ALK5, TGF beta receptor II, and GIPC. [5] In one study with mouse fibroblasts, the overexpression of endoglin resulted in a reduction of some ECM components, decreased cellular migration, a change in cellular morphology and intercellular cluster formation. [20]
Endoglin has been found to be an auxiliary receptor for the TGF-beta receptor complex. [16] It thus is involved in modulating a response to the binding of TGF-beta1, TGF-beta3, activin-A, BMP-2, BMP-7 and BMP-9. Beside TGF-beta signaling endoglin may have other functions. It has been postulated that endoglin is involved in the cytoskeletal organization affecting cell morphology and migration. [21] Endoglin has a role in the development of the cardiovascular system and in vascular remodeling. Its expression is regulated during heart development . Experimental mice without the endoglin gene die due to cardiovascular abnormalities. [21]
In humans endoglin may be involved in the autosomal dominant disorder known as hereditary hemorrhagic telangiectasia (HHT) type 1. [9] HHT is actually the first human disease linked to the TGF beta receptor complex. [22] This condition leads to frequent nose bleeds, telangiectases on skin and mucosa and may cause arteriovenous malformations in different organs including brain, lung, and liver.
Some mutations that lead to this disorder are: [22]
Endoglin levels have been found to be elevated in pregnant women who subsequently develop preeclampsia. [23]
The role of endoglin plays in angiogenesis [24] and the modulation of TGF beta receptor signaling, which mediates cellular localization, cellular migration, cellular morphology, cell proliferation, cluster formation, etc., makes endoglin an important player in tumor growth and metastasis. [25] [26] Being able to target and efficiently reduce or halt neoangiogenesis in tumors would prevent metastasis of primary cancer cells into other areas of the body. [25] Also, it has been suggested that endoglin can be used for tumor imaging and prognosis. [25]
The role of endoglin in cancer can be contradicting at times since it is needed for neoangiogenesis in tumors, which is needed for tumor growth and survival, yet the reduction in expression of endoglin has in many cancers correlated with a negative outcome of that cancer. [5] In breast cancer, for example, the reduction of the full form of endoglin, and the increase of the soluble form of endoglin correlate with metastasis of cancer cells. [27] The TGF beta receptor-endoglin complex relay contradicting signals from TGF beta as well. TGF beta can act as a tumor suppressor in the premalignant stage of the benign neoplasm by inhibiting its growth and inducing apoptosis. [5] However, once the cancer cells have gone through the Hallmarks of Cancer and lost inhibitory growth responses, TGF beta mediates cell invasion, angiogenesis (with the help of endoglin), immune system evasion, and their ECM composition, allowing them to become malignant. [5]
It has been shown that endoglin expression and TGF-beta secretion are attenuated in bone marrow stromal cells when they are cocultured with prostate cancer cells. [28] Also, the downstream TGF-beta/bone morphogenic protein (BMP) signaling pathway, which includes Smad1 and Smad2/3, were attenuated along with Smad-dependent gene transcription. [28] Another result in this study was that both Smad1/5/8-dependent inhibitor of DNA binding 1 expression and Smad2/3-dependent plasminogen activator inhibitor I had a reduction in expression and cell proliferation. [28] Ultimately, the cocultured prostate cancer cells altered the TGF-beta signaling in the bone stromal cells, which suggests this modulation is a mechanism of prostate cancer metastases facilitating their growth and survival in the reactive bone stroma. [28] This study emphasizes the importance of endoglin in TGF-beta signaling pathways in other cell types other than endothelial cells.
TRC105 is an experimental antibody targeted at endoglin as an anti-angiogenesis treatment for soft-tissue sarcoma. [29]
In cellular biology, paracrine signaling is a form of cell signaling, a type of cellular communication in which a cell produces a signal to induce changes in nearby cells, altering the behaviour of those cells. Signaling molecules known as paracrine factors diffuse over a relatively short distance, as opposed to cell signaling by endocrine factors, hormones which travel considerably longer distances via the circulatory system; juxtacrine interactions; and autocrine signaling. Cells that produce paracrine factors secrete them into the immediate extracellular environment. Factors then travel to nearby cells in which the gradient of factor received determines the outcome. However, the exact distance that paracrine factors can travel is not certain.
Vascular endothelial growth factor, originally known as vascular permeability factor (VPF), is a signal protein produced by many cells that stimulates the formation of blood vessels. To be specific, VEGF is a sub-family of growth factors, the platelet-derived growth factor family of cystine-knot growth factors. They are important signaling proteins involved in both vasculogenesis and angiogenesis.
An angiogenesis inhibitor is a substance that inhibits the growth of new blood vessels (angiogenesis). Some angiogenesis inhibitors are endogenous and a normal part of the body's control and others are obtained exogenously through pharmaceutical drugs or diet.
A co-receptor is a cell surface receptor that binds a signalling molecule in addition to a primary receptor in order to facilitate ligand recognition and initiate biological processes, such as entry of a pathogen into a host cell.
Perlecan (PLC) also known as basement membrane-specific heparan sulfate proteoglycan core protein (HSPG) or heparan sulfate proteoglycan 2 (HSPG2), is a protein that in humans is encoded by the HSPG2 gene. The HSPG2 gene codes for a 4,391 amino acid protein with a molecular weight of 468,829. It is one of the largest known proteins. The name perlecan comes from its appearance as a "string of pearls" in rotary shadowed images.
Mothers against decapentaplegic homolog 3 also known as SMAD family member 3 or SMAD3 is a protein that in humans is encoded by the SMAD3 gene.
SMAD4, also called SMAD family member 4, Mothers against decapentaplegic homolog 4, or DPC4 is a highly conserved protein present in all metazoans. It belongs to the SMAD family of transcription factor proteins, which act as mediators of TGF-β signal transduction. The TGFβ family of cytokines regulates critical processes during the lifecycle of metazoans, with important roles during embryo development, tissue homeostasis, regeneration, and immune regulation.
The transforming growth factor beta (TGFB) signaling pathway is involved in many cellular processes in both the adult organism and the developing embryo including cell growth, cell differentiation, cell migration, apoptosis, cellular homeostasis and other cellular functions. The TGFB signaling pathways are conserved. In spite of the wide range of cellular processes that the TGFβ signaling pathway regulates, the process is relatively simple. TGFβ superfamily ligands bind to a type II receptor, which recruits and phosphorylates a type I receptor. The type I receptor then phosphorylates receptor-regulated SMADs (R-SMADs) which can now bind the coSMAD SMAD4. R-SMAD/coSMAD complexes accumulate in the nucleus where they act as transcription factors and participate in the regulation of target gene expression.
Bone morphogenetic protein receptor type II or BMPR2 is a serine/threonine receptor kinase encoded by the BMPR2 gene. It binds bone morphogenetic proteins, members of the TGF beta superfamily of ligands, which are involved in paracrine signaling. BMPs are involved in a host of cellular functions including osteogenesis, cell growth and cell differentiation. Signaling in the BMP pathway begins with the binding of a BMP to the type II receptor. This causes the recruitment of a BMP type I receptor, which the type II receptor phosphorylates. The type I receptor phosphorylates an R-SMAD, a transcriptional regulator.
Thrombospondin 1, abbreviated as THBS1, is a protein that in humans is encoded by the THBS1 gene.
CTGF, also known as CCN2 or connective tissue growth factor, is a matricellular protein of the CCN family of extracellular matrix-associated heparin-binding proteins. CTGF has important roles in many biological processes, including cell adhesion, migration, proliferation, angiogenesis, skeletal development, and tissue wound repair, and is critically involved in fibrotic disease and several forms of cancers.
Betaglycan also known as Transforming growth factor beta receptor III (TGFBR3), is a cell-surface chondroitin sulfate / heparan sulfate proteoglycan >300 kDa in molecular weight. Betaglycan binds to various members of the TGF-beta superfamily of ligands via its core protein, and bFGF via its heparan sulfate chains. TGFBR3 is the most widely expressed type of TGF-beta receptor. Its affinity towards all individual isoforms of TGF-beta is similarly high and therefore it plays an important role as a coreceptor mediating the binding of TGF-beta to its other receptors - specifically TGFBR2. The intrinsic kinase activity of this receptor has not yet been described. In regard of TGF-beta signalling it is generally considered a non-signaling receptor or a coreceptor. By binding to various member of the TGF-beta superfamily at the cell surface it acts as a reservoir of TGF-beta.
Transforming growth factor beta receptor I is a membrane-bound TGF beta receptor protein of the TGF-beta receptor family for the TGF beta superfamily of signaling ligands. TGFBR1 is its human gene.
Transforming growth factor, beta receptor II (70/80kDa) is a TGF beta receptor. TGFBR2 is its human gene.
Growth differentiation factor 2 (GDF2) also known as bone morphogenetic protein (BMP)-9 is a protein that in humans is encoded by the GDF2 gene. GDF2 belongs to the transforming growth factor beta superfamily.
Syndecans are single transmembrane domain proteins that are thought to act as coreceptors, especially for G protein-coupled receptors. More specifically, these core proteins carry three to five heparan sulfate and chondroitin sulfate chains, i.e. they are proteoglycans, which allow for interaction with a large variety of ligands including fibroblast growth factors, vascular endothelial growth factor, transforming growth factor-beta, fibronectin and antithrombin-1. Interactions between fibronectin and some syndecans can be modulated by the extracellular matrix protein tenascin C.
Tenascin C (TN-C) is a glycoprotein that in humans is encoded by the TNC gene. It is expressed in the extracellular matrix of various tissues during development, disease or injury, and in restricted neurogenic areas of the central nervous system. Tenascin-C is the founding member of the tenascin protein family. In the embryo it is made by migrating cells like the neural crest; it is also abundant in developing tendons, bone and cartilage.
Integrin beta-6 is a protein that in humans is encoded by the ITGB6 gene. It is the β6 subunit of the integrin αvβ6. Integrins are αβ heterodimeric glycoproteins which span the cell’s membrane, integrating the outside and inside of the cell. Integrins bind to specific extracellular proteins in the extracellular matrix or on other cells and subsequently transduce signals intracellularly to affect cell behaviour. One α and one β subunit associate non-covalently to form 24 unique integrins found in mammals. While some β integrin subunits partner with multiple α subunits, β6 associates exclusively with the αv subunit. Thus, the function of ITGB6 is entirely associated with the integrin αvβ6.
Angiogenesis is the process of forming new blood vessels from existing blood vessels, formed in vasculogenesis. It is a highly complex process involving extensive interplay between cells, soluble factors, and the extracellular matrix (ECM). Angiogenesis is critical during normal physiological development, but it also occurs in adults during inflammation, wound healing, ischemia, and in pathological conditions such as rheumatoid arthritis, hemangioma, and tumor growth. Proteolysis has been indicated as one of the first and most sustained activities involved in the formation of new blood vessels. Numerous proteases including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase domain (ADAM), a disintegrin and metalloproteinase domain with throbospondin motifs (ADAMTS), and cysteine and serine proteases are involved in angiogenesis. This article focuses on the important and diverse roles that these proteases play in the regulation of angiogenesis.
Transforming growth factor beta (TGF-β) is a potent cell regulatory polypeptide homodimer of 25kD. It is a multifunctional signaling molecule with more than 40 related family members. TGF-β plays a role in a wide array of cellular processes including early embryonic development, cell growth, differentiation, motility, and apoptosis.
