Editorial: Role of membrane-bound and circulating endoglin in disease

4 p.Peer reviewe

The Role of the TGF-beta Coreceptor Endoglin in Cancer

Endoglin (CD105) is an auxiliary membrane receptor of transforming growth factor beta (TGF-beta) that interacts with type I and type II TGF-beta receptors and modulates TGF-beta signaling. Endoglin is overexpressed in the tumor-associated vascular endothelium, where it modulates angiogenesis. This feature makes endoglin a promising target for antiangiogenic cancer therapy. In addition, recent studies on human and experimental models of carcinogenesis point to an important tumor cell-autonomous role of endoglin by regulating proliferation, migration, invasion, and metastasis. These studies suggest that endoglin behaves as a suppressor of malignancy in experimental and human epithelial carcinogenesis, although it can also promote metastasis in other types of cancer. In this review, we evaluate the implication of endoglin in tumor development underlying studies developed in our laboratories in recent years

Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein.

Endoglin (Eng, CD105) is a type I membrane glycoprotein that functions in endothelial cells as an auxiliary receptor for transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) family members and as an integrin ligand, modulating the vascular pathophysiology. Besides the membrane-bound endoglin, there is a soluble form of endoglin (sEng) that can be generated by the action of the matrix metalloproteinase (MMP)-14 or -12 on the juxtamembrane region of its ectodomain. High levels of sEng have been reported in patients with preeclampsia, hypercholesterolemia, atherosclerosis and cancer. In addition, sEng is a marker of cardiovascular damage in patients with hypertension and diabetes, plays a pathogenic role in preeclampsia, and inhibits angiogenesis and tumor proliferation, migration, and invasion in cancer. However, the mechanisms of action of sEng have not yet been elucidated, and new tools and experimental approaches are necessary to advance in this field. To this end, we aimed to obtain a fluorescent form of sEng as a new tool for biological imaging. Thus, we cloned the extracellular domain of endoglin in the pEGFP-N1 plasmid to generate a fusion protein with green fluorescent protein (GFP), giving rise to pEGFP-N1/Eng.EC. The recombinant fusion protein was characterized by transient and stable transfections in CHO-K1 cells using fluorescence microscopy, SDS-PAGE, immunodetection, and ELISA techniques. Upon transfection with pEGFP-N1/Eng.EC, fluorescence was readily detected in cells, indicating that the GFP contained in the recombinant protein was properly folded into the cytosol. Furthermore, as evidenced by Western blot analysis, the secreted fusion protein yielded the expected molecular mass and displayed a specific fluorescent signal. The fusion protein was also able to bind to BMP9 and BMP10 in vitro. Therefore, the construct described here could be used as a tool for functional in vitro studies of the extracellular domain of endoglin

Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners

This article belongs to the Special Issue TGF-beta/BMP Signaling PathwayEndoglin is a 180-kDa glycoprotein receptor primarily expressed by the vascular endothelium and involved in cardiovascular disease and cancer. Heterozygous mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1, a vascular disease that presents with nasal and gastrointestinal bleeding, skin and mucosa telangiectases, and arteriovenous malformations in internal organs. A circulating form of endoglin (alias soluble endoglin, sEng), proteolytically released from the membrane-bound protein, has been observed in several inflammation-related pathological conditions and appears to contribute to endothelial dysfunction and cancer development through unknown mechanisms. Membrane-bound endoglin is an auxiliary component of the TGF-ß receptor complex and the extracellular region of endoglin has been shown to interact with types I and II TGF-ß receptors, as well as with BMP9 and BMP10 ligands, both members of the TGF-ß family. To search for novel protein interactors, we screened a microarray containing over 9000 unique human proteins using recombinant sEng as bait. We find that sEng binds with high affinity, at least, to 22 new proteins. Among these, we validated the interaction of endoglin with galectin-3, a secreted member of the lectin family with capacity to bind membrane glycoproteins, and with tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin-protein ligase. Using human endothelial cells and Chinese hamster ovary cells, we showed that endoglin co-immunoprecipitates and co-localizes with galectin-3 or TRIM21. These results open new research avenues on endoglin function and regulation.This work was supported by grants from Ministerio de Ciencia, Innovación y Universidades of Spain (SAF2013-43421-R to CB and SAF2017-84183-R to MQ), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 to CB and contract CNV-234-PRF-360 to LR-L) and Consejo Superior de Investigaciones Científicas (CSIC; 201920E022 to CB). JC-V was supported by a postdoctoral contract co-funded by Consejo Superior de Investigaciones Científicas, Ministerio de Ciencia, Innovación y Universidades and the European Social Fund (ESF). CIBERER and CIBERNED are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain supported by European Regional Development (FEDER) funds

Correlation Between Endoglin and Malignant Phenotype in Human Melanoma Cells: Analysis of hsa-mir-214 and hsa-mir-370 in Cells and Their Extracellular Vesicles.

Endoglin (CD105) is an auxiliary receptor of transforming growth factor (TGF)-β family members that is expressed in human melanomas. It is heterogeneously expressed by primary and metastatic melanoma cells, and endoglin targeting as a therapeutic strategy for melanoma tumors is currently been explored. However, its involvement in tumor development and malignancy is not fully understood. Here, we find that endoglin expression correlates with malignancy of primary melanomas and cultured melanoma cell lines. Next, we have analyzed the effect of ectopic endoglin expression on two miRNAs (hsa-mir-214 and hsa-mir-370), both involved in melanoma tumor progression and endoglin regulation. We show that compared with control cells, overexpression of endoglin in the WM-164 melanoma cell line induces; (i) a significant increase of hsa-mir-214 levels in small extracellular vesicles (EVs) as well as an increased trend in cells; and (ii) significantly lower levels of hsa-mir-370 in the EVs fractions, whereas no significant differences were found in cells. As hsa-mir-214 and hsa-mir-370 are not just involved in melanoma tumor progression, but they can also target endoglin-expressing endothelial cells in the tumor vasculature, these results suggest a complex and differential regulatory mechanism involving the intracellular and extracellular signaling of hsa-mir-214 and hsa-mir-370 in melanoma development and progression.S

5'UTR mutations of ENG cause hereditary hemorrhagic telangiectasia

<p>Abstract</p> <p>Background</p> <p>Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder characterized by epistaxis, arteriovenous malformations, and telangiectases. The majority of the patients have a mutation in the coding region of the activin A receptor type II-like 1 (<it>ACVRL1</it>) or Endoglin (<it>ENG</it>) gene. However, in approximately 15% of cases, sequencing analysis and deletion/duplication testing fail to identify mutations in the coding regions of these genes. Knowing its vital role in transcription and translation control, we were prompted to investigate the 5'untranslated region (UTR) of <it>ENG</it>.</p> <p>Methods and Results</p> <p>We sequenced the 5'UTR of <it>ENG </it>for 154 HHT patients without mutations in <it>ENG </it>or <it>ACVRL1 </it>coding regions. We found a mutation (c.-127C > T), which is predicted to affect translation initiation and alter the reading frame of endoglin. This mutation was found in a family with linkage to the <it>ENG</it>, as well as in three other patients, one of which had an affected sibling with the same mutation. <it>In vitro </it>expression studies showed that a construct with the c.-127C > T mutation alters the translation and decreases the level of the endoglin protein. In addition, a c.-9G > A mutation was found in three patients, one of whom was homozygous for this mutation. Expression studies showed decreased protein levels suggesting that the c.-9G > A is a hypomorphic mutation.</p> <p>Conclusions</p> <p>Our results emphasize the need for the inclusion of the 5'UTR region of <it>ENG </it>in clinical testing for HHT.</p

Mutation study of Spanish patients with Hereditary Hemorrhagic Telangiectasia

<p>Abstract</p> <p>Background</p> <p>Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant and age-dependent vascular disorder characterised mainly by mutations in the Endoglin (ENG) or activin receptor-like kinase-1 (ALK1, ACVRL1) genes.</p> <p>Methods</p> <p>Here, we have identified 22 ALK1 mutations and 15 ENG mutations, many of which had not previously been reported, in independent Spanish families afflicted with HHT.</p> <p>Results</p> <p>We identified mutations in thirty-seven unrelated families. A detailed analysis of clinical symptoms was recorded for each patient analyzed, with a higher significant presence of pulmonary arteriovenous malformations (PAVM) in HHT1 patients over HHT2. Twenty-two mutations in ALK1 and fifteen in ENG genes were identified. Many of them, almost half, represented new mutations in ALK1 and in ENG. Missense mutations in ENG and ALK1 were localized in a tridimensional protein structure model.</p> <p>Conclusion</p> <p>Overall, ALK1 mutations (HHT2) were predominant over ENG mutations (HHT1) in our Spanish population, in agreement with previous data from our country and other Mediterranean countries (France, Italy), but different to Northern Europe or North America. There was a significant increase of PAVM associated with HHT1 over HHT2 in these families.</p

Characterization of the human Activin-A receptor type II-like kinase 1 (ACVRL1) promoter and its regulation by Sp1

<p>Abstract</p> <p>Background</p> <p>Activin receptor-like kinase 1 (ALK1) is a Transforming Growth Factor-β (TGF-β) receptor type I, mainly expressed in endothelial cells that plays a pivotal role in vascular remodelling and angiogenesis. Mutations in the ALK1 gene (<it>ACVRL1</it>) give rise to Hereditary Haemorrhagic Telangiectasia, a dominant autosomal vascular dysplasia caused by a haploinsufficiency mechanism. In spite of its patho-physiological relevance, little is known about the transcriptional regulation of <it>ACVRL1</it>. Here, we have studied the different origins of <it>ACVRL1 </it>transcription, we have analyzed <it>in silico </it>its 5'-proximal promoter sequence and we have characterized the role of Sp1 in the transcriptional regulation of <it>ACVRL1</it>.</p> <p>Results</p> <p>We have performed a 5'Rapid Amplification of cDNA Ends (5'RACE) of <it>ACVRL1 </it>transcripts, finding two new transcriptional origins, upstream of the one previously described, that give rise to a new exon undiscovered to date. The 5'-proximal promoter region of <it>ACVRL1 </it>(-1,035/+210) was analyzed <it>in silico</it>, finding that it lacks TATA/CAAT boxes, but contains a remarkably high number of GC-rich Sp1 consensus sites. In cells lacking Sp1, <it>ACVRL1 </it>promoter reporters did not present any significant transcriptional activity, whereas increasing concentrations of Sp1 triggered a dose-dependent stimulation of its transcription. Moreover, silencing Sp1 in HEK293T cells resulted in a marked decrease of <it>ACVRL1 </it>transcriptional activity. Chromatin immunoprecipitation assays demonstrated multiple Sp1 binding sites along the proximal promoter region of <it>ACVRL1 </it>in endothelial cells. Furthermore, demethylation of CpG islands, led to an increase in <it>ACVRL1 </it>transcription, whereas <it>in vitro </it>hypermethylation resulted in the abolishment of Sp1-dependent transcriptional activation of <it>ACVRL1</it>.</p> <p>Conclusions</p> <p>Our results describe two new transcriptional start sites in <it>ACVRL1 </it>gene, and indicate that Sp1 is a key regulator of <it>ACVRL1 </it>transcription, providing new insights into the molecular mechanisms that contribute to the expression of <it>ACVRL1 </it>gene. Moreover, our data show that the methylation status of CpG islands markedly modulates the Sp1 regulation of <it>ACVRL1 </it>gene transcriptional activity.</p

TNFα down-regulates CD105 expression in vascular endothelial cells: a comparative study with TGFß1

The vascular endothelium participates in angiogenesis, inflammation and the immune response, which are modulated by vasoactive cytokines such as tumour necrosis factor-α (TNFα) and transforming growth factor-ß1 (TGFß1). CD105 is a component of the TGFß receptor complex and is abundantly expressed in activated/injured endothelium where it is implicated in multiple cellular processes. Up-regulation of CD105 in synovial cells of rheumatoid arthritis and psoriatic lesions implies a possible role in the pathogenesis of such inflammatory disorders. The pro-inflammatory cytokine, TNFα, and anti-inflammatory cytokine, TGFß1, regulate multiple cellular processes such as proliferation, differentiation and apoptosis. Our hypothesis is that CD105 gene expression in endothelial cells is regulated by the multifunctional cytokines TNFα and TGFß1. By using human dermal microvascular endothelial cells the present study has shown that long-term treatment with TNFα (0.1-5 ng/ml) elicited a concentration- and time-dependent significant suppression (over 50% reduction) in CD105 protein levels. The observations that no significant alterations in the CD105 mRNA levels or in the CD105 promoter activity were found and that the potent inhibitor of NFÎB, PDTC, did not affect the TNFα action suggest that CD105 down-regulation by TNF· is not at the transcriptional level. In contrast to TNFα, TGFß1 significantly elevated CD105 protein and mRNA expression (<2-fold increase) through activation of its promoter activity. From these data we conclude that TNFα and TGFß1 exert opposing effects on CD105 expression in human vascular endothelial cells and that CD105 is enmeshed in the network of signal pathways modulating multiple cellular functions

CD105 prevents apoptosis in hypoxic endothelial cells

CD105, a marker of endothelial cells, is abundantly expressed in tissues undergoing angiogenesis and is a receptor for transforming growth factorß. The pivotal role of CD105 in the vascular system was demonstrated by the severe vascular defects that occur in CD105-knockout mice, but the exact mechanisms for CD105 regulation of vascular development have not been fully elucidated. In light of the function of CD105 and the importance of hypoxia in neovascularisation, we speculated that CD105 is involved in hypoxia-initiated angiogenesis. Using tissue-cultured human microvascular endothelial cells, we have investigated the effects of hypoxic stress on CD105 gene expression. Hypoxia induced a significant increase in membrane-bound and secreted CD105 protein levels. CD105 mRNA and promoter activity were also markedly elevated, the latter returning to the basal level after 16 hours of hypoxic stress. Hypoxia induced cell cycle arrest at the G0/G1 phases and massive cell apoptosis after 24 hours through a reduction in the Bcl-2 to Bax ratio, downregulation of Bcl-XL and Mcl-1, and upregulation of caspase-3 and caspase-8. The consequence of CD105 upregulation was revealed using an antisense approach and a TUNEL assay. Suppression of CD105 increased cell apoptosis under hypoxic stress in the absence of TGFß1. Furthermore, hypoxia and TGFß1 synergistically induced apoptosis in the CD105-deficient cells but not in the control cells. We conclude that hypoxia is a potent stimulus for CD105 gene expression in vascular endothelial cells, which in turn attenuates cell apoptosis and thus contributes to angiogenesis