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Resumen del trabajo presentado al 38th Annual Meeting of the European Thyroid Association celebrado en Santiago de Compostela (España) del 6 al 10 de septiembre de 2014., Transforming growth factor beta (TGFβ), which plays a key role in cancer and fibrotic disorders, mediates its actions mainly through activation of Smad transcription factors, which bind to Smad Binding Elements (SBEs) in target genes. We have previously observed that the thyroid hormone T3 blocks transactivation of SBE-containing reporter plasmids by TGFβ, and represses transcription of endogenous TGF target genes. We have now analysed the thyroid hormone receptor (TR) isoform as well as the receptor domains responsible for this transcriptional antagonism. In GH4C1 pituitary cells, the TR specific agonist GC1 is as potent as T3 to repress SMAD-dependent transactivation. However, expression of TR in other cell types also mediates repression of TGFβ-dependent transactivation by T3. Using TR and TR mutants we have observed that the DNA binding domain (DBD) is essential for the repressive effect, whereas the ligand-dependent transcriptional activation domain (AF-2), responsible for coactivators binding, appears to be dispensable. Moreover, we have detected a direct interaction of TRα and TRß with Smad2/3 and Smad4, which is reversed by T3. Mutations in the AF-2 domain do no alter association with Smads, while the DBD plays an important role in this interaction. In chromatin immunoprecipitation (ChIP) assays, we have observed TR recruitment to SBE-containing regions. T3 inhibits this association, as well as TGFß-dependent recruitment of Smads and coactivators such as p300. In agreement with the transcriptional cross-talk between T3 and TGFß signaling, hyperthyroidism alleviates the fibrotic response induced by bleomycin in mice skin. In addition, hyperthyroidism decreases Smad2/3 phosphorylation and hepatocyte necrosis in a model of acute carbon tetrachloride administration, reducing liver fibrosis and collagen deposition after chronic administration of this compound. These results demonstrate that TRs, in a ligand-dependent manner, antagonize TGF responses in cultured cells and appear to play a novel role as suppressors of fibrotic responses in vivo.

13 p.-3 fig.-1 tab., Background: Disrupted endothelial BMP9/10 signaling may contribute to the pathophysiology of both hereditary hemorrhagic telangiectasia (HHT) and pulmonary arterial hypertension (PAH), yet loss of circulating BMP9 has not been confirmed in individuals with ultra-rare homozygous GDF2 (BMP9 gene) nonsense mutations. We studied two pediatric patients homozygous for GDF2 (BMP9 gene) nonsense mutations: one with PAH (c.[76C>T];[76C>T] or p.[Gln26Ter];[Gln26Ter] and a new individual with pulmonary arteriovenous malformations (PAVMs; c.[835G>T];[835G>T] or p.[Glu279Ter];[Glu279Ter]); both with facial telangiectases., Methods: Plasma samples were assayed for BMP9 and BMP10 by ELISA. In parallel, serum BMP activity was assayed using an endothelial BRE-luciferase reporter cell line (HMEC1-BRE). Proteins were expressed for assessment of secretion and processing., Results: Plasma levels of both BMP9 and BMP10 were undetectable in the two homozygous index cases and this corresponded to low serum-derived endothelial BMP activity in the patients. Measured BMP9 and BMP10 levels were reduced in the asymptomatic heterozygous p.[Glu279Ter] parents, but serum activity was normal. Although expression studies suggested alternate translation can be initiated at Met57 in the p.[Gln26Ter] mutant, this does not result in secretion of functional BMP9., Conclusion: Collectively, these data show that homozygous GDF2 mutations, leading to a loss of circulating BMP9 and BMP10, can cause either pediatric PAH and/or "HHT-like" telangiectases and PAVMs. Although patients reported to date have manifestations that overlap with those of HHT, none meet the Curaçao criteria for HHT and seem distinct from HHT in terms of the location and appearance of telangiectases, and a tendency for tiny, diffuse PAVMs., This study was supported by British Heart Foundation Grant/Award Numbers: FS/15/62/323032, RG/13/4/30107, and RG/19/3/34265; Consejo Superior de Investigaciones Científicas (CSIC) Grant/Award Number: 201920E022; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) Grant/ Award Numbers: ISCIII-CB06/07/0038 and CNV-234-PRF-360.

13 p.-5 fig., 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., This research was funded by grants from Ministerio de Ciencia, Innovación y Universidades of Spain (SAF2013-43421-R to C.B. and RTI2018-102242-B-I00 to M.C.V.), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 to C.B. and contract CNV-234-PRF-360 to L.R.-L.) and Consejo Superior de Investigaciones Científicas (CSIC; 201920E022 to C.B.) and the British Heart Foundation (Programme Grant RG/19/3/34265 to P.D.U. and N.W.M.). CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.

7 p.-4 fig., Hereditary hemorrhagic telangiectasia (HHT) is a vascular rare disease characterized by nose and gastrointestinal bleeding, skin and mucosa telangiectasias, and arteriovenous malformations in internal organs. HHT shows an autosomal dominant inheritance and a worldwide prevalence of approximately 1:5000 individuals. In >80% of patients, HHT is caused by mutations in either ENG (HHT1) or ACVRL1 (HHT2) genes, which code for the membrane proteins Endoglin and Activin A Receptor Type II-Like Kinase 1 (ALK1), respectively, both belonging to the TGF-β/BMP signaling pathway. In this work, we describe a novel mutation in exon 9 of ENG (c.1145 G > A) found in five affected members of a family, all of them with characteristic symptoms of HHT. This mutation involves Cys382 residue of the Endoglin protein (p.Cys382 > Tyr) in the zona pellucida (ZP) module of its extracellular region. This is a critical residue involved in a conserved intrachain disulphide bond and in the correct folding of the protein. In fact, transfection studies in human cells using Endoglin expression vectors demonstrated that the p.Cys382 > Tyr mutation results in a marked reduction in the levels of the Endoglin protein. These results demonstrate the pathogenic role for this variant in HHT1 and confirm the key function of Cys382 in Endoglin expression., This work was supported by the Ministerio de Economía, Industria y Competitividad (Grant SAF2013-43421-R to CB); the Consejo Superior de Investigaciones Científicas (Grant 201420E039 and 201920E022 to CB); and the Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER; Grant ISCIII-CB06/07/0038 to CB and contract to LR-L) of Spain. CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.

7 p.-4 fig., Hereditary hemorrhagic telangiectasia (HHT) is a vascular disease characterized by nose and gastrointestinal bleeding, telangiectases in skin and mucosa, and arteriovenous malformations in major internal organs. Most patients carry a mutation in the coding region of the endoglin (ENG) or activin A receptor type II-1 (ACVRL1) gene. Nonetheless, in around 15% of patients, sequencing analysis and duplication/deletion tests fail to pinpoint mutations in the coding regions of these genes. In these cases, it has been shown that sequencing of the 5'-untranslated region (5'UTR) of ENG may be useful to identify novel mutations in the ENG non-coding region. Here we report the genetic characterization and functional analysis of the heterozygous mutation c.-142A>T in the 5'UTR region of ENG found in a family with several members affected by HHT. This variant gives rise to a new initiation codon of the protein that involves the change in its open reading frame. Transfection studies in monkey cells using endoglin expression vectors demonstrated that c-142A>T mutation results in a clear reduction in the levels of the endoglin protein. These results support the inclusion of the 5'UTR of ENG in the standard genetic testing for HHT to increase its sensitivity., This work was supported by grants from Ministerio de Economia, Industria y Competitividad (SAF2013-43421-R to CB), Mobility Program Salvador de Madariaga, Ministerio de Educacion, Cultura y Deporte (PRX17/00142 to CB), Consejo Superior de Investigaciones Cientificas (201420E039 and 201920E022 to CB), and Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 to CB and contract CNV-234-PRF-360 to LR-L) of Spain.

20 p.-8 fig.-1 tab., 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., This work was supported by grants from Ministerio de Ciencia, Innovación y Universidades (SAF2013-43421-R to CB), Consejo Superior de Investigaciones Científicas (201920E022 to CB), Fondo de Investigación Sanitaria (FIS) de la Seguridad Social (PI-20/01553 to JLR-P), and 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) of Spain. CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds. The CNIO, certified as a Severo Ochoa Excellence Centre, is supported by the Spanish government through the ISCIII.