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1.
Cancers (Basel) ; 13(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204675

RESUMO

Angiogenesis, i.e., the formation of new blood vessels from pre-existing endothelial cell (EC)-lined vessels, is critical for tissue development and also contributes to neovascularization-related diseases, such as cancer. Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are among many secreted cytokines that regulate EC function. While several pharmacological anti-angiogenic agents have reached the clinic, further improvement is needed to increase clinical efficacy and to overcome acquired therapy resistance. More insights into the functional consequences of targeting specific pathways that modulate blood vessel formation may lead to new therapeutic approaches. Here, we synthesized and identified two macrocyclic small molecular compounds termed OD16 and OD29 that inhibit BMP type I receptor (BMPRI)-induced SMAD1/5 phosphorylation and downstream gene expression in ECs. Of note, OD16 and OD29 demonstrated higher specificity against BMPRI activin receptor-like kinase 1/2 (ALK1/2) than the commonly used small molecule BMPRI kinase inhibitor LDN-193189. OD29, but not OD16, also potently inhibited VEGF-induced extracellular regulated kinase MAP kinase phosphorylation in ECs. In vitro, OD16 and OD29 exerted strong inhibition of BMP9 and VEGF-induced ECs migration, invasion and cord formation. Using Tg (fli:EGFP) zebrafish embryos, we found that OD16 and OD29 potently antagonized dorsal longitudinal anastomotic vessel (DLAV), intra segmental vessel (ISV), and subintestinal vessel (SIV) formation during embryonic development. Moreover, the MDA-MB-231 breast cancer cell-induced tumor angiogenesis in zebrafish embryos was significantly decreased by OD16 and OD29. Both macrocyclic compounds might provide a steppingstone for the development of novel anti-angiogenesis therapeutic agents.

2.
Front Cell Dev Biol ; 9: 616610, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33644053

RESUMO

Endothelial-to-mesenchymal transition (EndMT) plays an important role in embryonic development and disease progression. Yet, how different members of the transforming growth factor-ß (TGF-ß) family regulate EndMT is not well understood. In the current study, we report that TGF-ß2, but not bone morphogenetic protein (BMP)9, triggers EndMT in murine endothelial MS-1 and 2H11 cells. TGF-ß2 strongly upregulates the transcription factor SNAIL, and the depletion of Snail is sufficient to abrogate TGF-ß2-triggered mesenchymal-like cell morphology acquisition and EndMT-related molecular changes. Although SLUG is not regulated by TGF-ß2, knocking out Slug also partly inhibits TGF-ß2-induced EndMT in 2H11 cells. Interestingly, in addition to SNAIL and SLUG, BMP9 stimulates inhibitor of DNA binding (ID) proteins. The suppression of Id1, Id2, or Id3 expression facilitated BMP9 in inducing EndMT and, in contrast, ectopic expression of ID1, ID2, or ID3 abrogated TGF-ß2-mediated EndMT. Altogether, our results show that SNAIL is critical and indispensable for TGF-ß2-mediated EndMT. Although SLUG is also involved in the EndMT process, it plays less of a crucial role in it. In contrast, ID proteins are essential for maintaining endothelial traits and repressing the function of SNAIL and SLUG during the EndMT process. These data suggest that the control over endothelial vs. mesenchymal cell states is determined, at least in part, by a balance between the expression of SNAIL/SLUG and ID proteins.

3.
Proc Natl Acad Sci U S A ; 117(47): 29684-29690, 2020 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-33184177

RESUMO

Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein-protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor ([Formula: see text] Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein-protein interaction involved in actin filament processing and cell migration.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Moléculas de Adesão Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Células Jurkat , Prolina/metabolismo , Ligação Proteica/efeitos dos fármacos , Peixe-Zebra
4.
J Vis Exp ; (164)2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33191940

RESUMO

Transforming growth factor-ß (TGF-ß) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-ß signaling can lead to breast cancer. TGF-ß elicits its effects on proliferation and differentiation via specific cell surface TGF-ß type I and type II receptors (i.e., TßRI and TßRII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-ß-induced heteromeric complex formation, activated TßRI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-ß acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-ß signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-ß-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-ß-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-ß receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-ß-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-ß-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Benzamidas/farmacologia , Dioxóis/farmacologia , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Pirazóis/farmacologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Transcrição Genética/efeitos dos fármacos
5.
J Mol Cell Biol ; 12(2): 138-151, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31291647

RESUMO

The transforming growth factor-ß (TGF-ß) family controls embryogenesis, stem cell differentiation, and tissue homeostasis. However, how post-translation modifications contribute to fine-tuning of TGF-ß family signaling responses is not well understood. Inhibitory (I)-Smads can antagonize TGF-ß/Smad signaling by recruiting Smurf E3 ubiquitin ligases to target the active TGF-ß receptor for proteasomal degradation. A proteomic interaction screen identified Vpr binding protein (VprBP) as novel binding partner of Smad7. Mis-expression studies revealed that VprBP negatively controls Smad2 phosphorylation, Smad2-Smad4 interaction, as well as TGF-ß target gene expression. VprBP was found to promote Smad7-Smurf1-TßRI complex formation and induce proteasomal degradation of TGF-ß type I receptor (TßRI). Moreover, VprBP appears to stabilize Smurf1 by suppressing Smurf1 poly-ubiquitination. In multiple adult and mouse embryonic stem cells, depletion of VprBP promotes TGF-ß or Activin-induced responses. In the mouse embryo VprBP expression negatively correlates with mesoderm marker expression, and VprBP attenuated mesoderm induction during zebrafish embryogenesis. Our findings thereby uncover a novel regulatory mechanism by which Smurf1 controls the TGF-ß and Activin cascade and identify VprBP as a critical determinant of embryonic mesoderm induction.


Assuntos
Ativinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ativinas/farmacologia , Animais , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HaCaT , Células Hep G2 , Humanos , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteína Smad7/genética , Proteína Smad7/metabolismo , Transfecção , Fator de Crescimento Transformador beta/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Peixe-Zebra/embriologia
6.
Clin Cancer Res ; 26(6): 1460-1473, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31857432

RESUMO

PURPOSE: Therapies directed to specific molecular targets are still unmet for patients with triple-negative breast cancer (TNBC). Deubiquitinases (DUB) are emerging drug targets. The identification of highly active DUBs in TNBC may lead to novel therapies. EXPERIMENTAL DESIGN: Using DUB activity probes, we profiled global DUB activities in 52 breast cancer cell lines and 52 patients' tumor tissues. To validate our findings in vivo, we employed both zebrafish and murine breast cancer xenograft models. Cellular and molecular mechanisms were elucidated using in vivo and in vitro biochemical methods. A specific inhibitor was synthesized, and its biochemical and biological functions were assessed in a range of assays. Finally, we used patient sera samples to investigate clinical correlations. RESULTS: Two DUB activity profiling approaches identified UCHL1 as being highly active in TNBC cell lines and aggressive tumors. Functionally, UCHL1 promoted metastasis in zebrafish and murine breast cancer xenograft models. Mechanistically, UCHL1 facilitates TGFß signaling-induced metastasis by protecting TGFß type I receptor and SMAD2 from ubiquitination. We found that these responses are potently suppressed by the specific UCHL1 inhibitor, 6RK73. Furthermore, UCHL1 levels were significantly increased in sera of patients with TNBC, and highly enriched in sera exosomes as well as TNBC cell-conditioned media. UCHL1-enriched exosomes stimulated breast cancer migration and extravasation, suggesting that UCHL1 may act in a paracrine manner to promote tumor progression. CONCLUSIONS: Our DUB activity profiling identified UCHL1 as a candidate oncoprotein that promotes TGFß-induced breast cancer metastasis and may provide a potential target for TNBC treatment.


Assuntos
Biomarcadores Tumorais/sangue , Enzimas Desubiquitinantes/metabolismo , Proteínas Oncogênicas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ubiquitina Tiolesterase/metabolismo , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Proteínas Oncogênicas/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/genética , Ubiquitina Tiolesterase/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
7.
Proc Natl Acad Sci U S A ; 116(36): 17800-17808, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31431534

RESUMO

Endoglin (ENG) is a coreceptor of the transforming growth factor-ß (TGFß) family signaling complex, which is highly expressed on endothelial cells and plays a key role in angiogenesis. Its extracellular domain can be cleaved and released into the circulation as soluble ENG (sENG). High circulating levels of sENG contribute to the pathogenesis of preeclampsia (PE). Circulating bone morphogenetic protein 9 (BMP9), a vascular quiescence and endothelial-protective factor, binds sENG with high affinity, but how sENG participates in BMP9 signaling complexes is not fully resolved. sENG was thought to be a ligand trap for BMP9, preventing type II receptor binding and BMP9 signaling. Here we show that, despite cell-surface ENG being a dimer linked by disulfide bonds, sENG purified from human placenta and plasma from PE patients is primarily in a monomeric form. Incubating monomeric sENG with the circulating form of BMP9 (prodomain-bound form) in solution leads to the release of the prodomain and formation of a sENG:BMP9 complex. Furthermore, we demonstrate that binding of sENG to BMP9 does not inhibit BMP9 signaling. Indeed, the sENG:BMP9 complex signals with comparable potency and specificity to BMP9 on human primary endothelial cells. The full signaling activity of the sENG:BMP9 complex required transmembrane ENG. This study confirms that rather than being an inhibitory ligand trap, increased circulating sENG might preferentially direct BMP9 signaling via cell-surface ENG at the endothelium. This is important for understanding the role of sENG in the pathobiology of PE and other cardiovascular diseases.


Assuntos
Endoglina/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Proteínas da Gravidez/metabolismo , Transdução de Sinais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Placenta/patologia , Pré-Eclâmpsia/patologia , Gravidez
8.
J Pathol ; 249(3): 356-367, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31257577

RESUMO

Pulmonary arterial hypertension (PAH) is characterised by an increase in mean pulmonary arterial pressure which almost invariably leads to right heart failure and premature death. More than 70% of familial PAH and 20% of idiopathic PAH patients carry heterozygous mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2). However, the incomplete penetrance of BMPR2 mutations suggests that other genetic and environmental factors contribute to the disease. In the current study, we investigate the contribution of autophagy in the degradation of BMPR2 in pulmonary vascular cells. We demonstrate that endogenous BMPR2 is degraded through the lysosome in primary human pulmonary artery endothelial (PAECs) and smooth muscle cells (PASMCs): two cell types that play a key role in the pathology of the disease. By means of an elegant HaloTag system, we show that a block in lysosomal degradation leads to increased levels of BMPR2 at the plasma membrane. In addition, pharmacological or genetic manipulations of autophagy allow us to conclude that autophagy activation contributes to BMPR2 degradation. It has to be further investigated whether the role of autophagy in the degradation of BMPR2 is direct or through the modulation of the endocytic pathway. Interestingly, using an iPSC-derived endothelial cell model, our findings indicate that BMPR2 heterozygosity alone is sufficient to cause an increased autophagic flux. Besides BMPR2 heterozygosity, pro-inflammatory cytokines also contribute to an augmented autophagy in lung vascular cells. Furthermore, we demonstrate an increase in microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) levels in lung sections from PAH induced in rats. Accordingly, pulmonary microvascular endothelial cells (MVECs) from end-stage idiopathic PAH patients present an elevated autophagic flux. Our findings support a model in which an increased autophagic flux in PAH patients contributes to a greater decrease in BMPR2 levels. Altogether, this study sheds light on the basic mechanisms of BMPR2 degradation and highlights a crucial role for autophagy in PAH. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Assuntos
Autofagia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Células Endoteliais/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Pressão Arterial , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/patologia , Feminino , Heterozigoto , Humanos , Mediadores da Inflamação/metabolismo , Lisossomos/metabolismo , Lisossomos/patologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , Proteólise , Hipertensão Arterial Pulmonar/patologia , Hipertensão Arterial Pulmonar/fisiopatologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Ratos , Transdução de Sinais , Adulto Jovem
9.
EMBO J ; 36(11): 1623-1639, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28468752

RESUMO

SMAD4 is a common intracellular effector for TGF-ß family cytokines, but the mechanism by which its activity is dynamically regulated is unclear. We demonstrated that ubiquitin-specific protease (USP) 4 strongly induces activin/BMP signaling by removing the inhibitory monoubiquitination from SMAD4. This modification was triggered by the recruitment of the E3 ligase, SMURF2, to SMAD4 following ligand-induced regulatory (R)-SMAD-SMAD4 complex formation. Whereas the interaction of the negative regulator c-SKI inhibits SMAD4 monoubiquitination, the ligand stimulates the recruitment of SMURF2 to the c-SKI-SMAD2 complex and triggers c-SKI ubiquitination and degradation. Thus, SMURF2 has a role in termination and initiation of TGF-ß family signaling. An increase in monoubiquitinated SMAD4 in USP4-depleted mouse embryonic stem cells (mESCs) decreased both the BMP- and activin-induced changes in the embryonic stem cell fate. USP4 sustained SMAD4 activity during activin- and BMP-mediated morphogenic events in early zebrafish embryos. Moreover, zebrafish depleted of USP4 exhibited defective cell migration and slower coordinated cell movement known as epiboly, both of which could be rescued by SMAD4. Therefore, USP4 is a critical determinant of SMAD4 activity.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Subunidades beta de Inibinas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteína Smad4/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Animais , Diferenciação Celular , Movimento Celular , Células Cultivadas , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina , Peixe-Zebra/embriologia
10.
J Biol Chem ; 292(10): 4099-4112, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28115518

RESUMO

The intensity and duration of TGF-ß signaling determine the cellular biological response. How this is negatively regulated is not well understood. Here, we identified a novel negative regulator of TGF-ß signaling, transmembrane p24-trafficking protein 10 (TMED10). TMED10 disrupts the complex formation between TGF-ß type I (also termed ALK5) and type II receptors (TßRII). Misexpression studies revealed that TMED10 attenuated TGF-ß-mediated signaling. A 20-amino acid-long region from Thr91 to Glu110 within the extracellular region of TMED10 was found to be crucial for TMED10 interaction with both ALK5 and TßRII. Synthetic peptides corresponding to this region inhibit both TGF-ß-induced Smad2 phosphorylation and Smad-dependent transcriptional reporter activity. In a xenograft cancer model, where previously TGF-ß was shown to elicit tumor-promoting effects, gain-of-function and loss-of-function studies for TMED10 revealed a decrease and increase in the tumor size, respectively. Thus, we determined herein that TMED10 expression levels are the key determinant for efficiency of TGF-ß receptor complex formation and signaling.


Assuntos
Neoplasias Mamárias Animais/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Serina-Treonina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Fator de Crescimento Transformador beta/genética , Proteínas de Transporte Vesicular/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Methods Mol Biol ; 1344: 35-47, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26520116

RESUMO

Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine that signals via transmembrane TGF-ß type I and type II serine/threonine kinases receptors, i.e., TßRI and TßRII. Upon TGF-ß-induced receptor complex formation, the TßRII kinase phosphorylates TßRI. Subsequently, the activated TßRI induces the phosphorylation of receptor regulated SMAD2 and SMAD3, which can form heteromeric complexes with Smad4. These heteromeric SMAD complexes accumulate in the nucleus, where they regulate target gene expression. The stability and membrane localization of TßRI is an important determinant to control the intensity and duration of TGF-ß signaling. TßRI is targeted for poly-ubiquitylation-mediated proteasomal degradation by the SMAD7-SMURF E3 ligase complex. We recently identified another important regulatory factor that controls TßRI levels in the cell membrane. As a strong inducer of TGF-ß signaling, ubiquitin-specific protease (USP) 4 was found to directly interact with TßRI and act as a deubiquitylating enzyme, thereby stabilizing TßRI levels at the plasma membrane. This chapter introduces methods for examining cell membrane receptor (TßRI) levels.


Assuntos
Membrana Celular/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Animais , Linhagem Celular , Humanos , Marcação por Isótopo , Ligantes , Proteínas de Membrana/metabolismo , Coloração e Rotulagem/métodos , Fator de Crescimento Transformador beta/metabolismo
12.
Gastroenterology ; 148(7): 1427-37.e8, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25736321

RESUMO

BACKGROUND & AIMS: Most colorectal cancer (CRC) cells with high levels of microsatellite instability (MSI-H) accumulate mutations at a microsatellite sequence in the gene encoding transforming growth factor ß receptor II (TGFBR2). TGFß signaling therefore is believed to be defective in these tumors, although CRC cells with TGFBR2 mutations have been reported to remain sensitive to TGFß. We investigated how TGFß signaling might continue in MSI-H CRC cells. METHODS: We sequenced the 10-adenines microsatellite sequence in the TGFBR2 gene of 32 MSI-H colon cancer tissues and 6 cell lines (HCT116, LS180, LS411N, RKO, SW48, and SW837). Activation of TGFß signaling was detected by SMAD2 phosphorylation and through use of a TGFß-responsive reporter construct in all CRC cell lines. Transcripts of TGFBR2 were knocked-down in CRC cells using short hairpin RNA. Full-length and mutant forms of TGFBR2 were expressed in LS411N cells, which do not respond to TGFß, and their activities were measured. RESULTS: SMAD2 was phosphorylated in most MSI-H CRC tissues (strong detection in 44% and weak detection in 34% of MSI-H tumors). Phosphorylation of SMAD2 in MSI-H cells required TGFBR2­even the form encoding a frameshift mutation. Transcription and translation of TGFBR2 with a 1-nucleotide deletion at its microsatellite sequence still produced a full-length TGFBR2 protein. However, protein expression required preservation of the TGFBR2 microsatellite sequence; cells in which this sequence was replaced with a synonymous nonmicrosatellite sequence did not produce functional TGFBR2 protein. CONCLUSION: TGFß signaling remains active in some MSI-H CRC cells despite the presence of frameshift mutations in the TGFBR2 gene because the mutated gene still expresses a functional protein. Strategies to reactivate TGFß signaling in colorectal tumors might not be warranted, and the functional effects of mutations at other regions of microsatellite instability should be evaluated.


Assuntos
Neoplasias Colorretais/genética , Mutação da Fase de Leitura , Instabilidade de Microssatélites , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/agonistas , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Células HCT116 , Células HEK293 , Humanos , Fenótipo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad2/genética , Proteína Smad2/metabolismo , Transcrição Genética , Transfecção
13.
Sci Signal ; 7(335): rs5, 2014 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-25056879

RESUMO

Transforming growth factor-ß (TGF-ß) signaling promotes cell motility by inducing epithelial-to-mesenchymal transitions (EMTs) in normal physiology and development, as well as in pathological conditions, such as cancer. We performed a time-resolved analysis of the proteomic and phosphoproteomic changes of cultured human keratinocytes undergoing EMT and cell cycle arrest in response to stimulation with TGF-ß. We quantified significant changes in 2079 proteins and 2892 phosphorylation sites regulated by TGF-ß. We identified several proteins known to be involved in TGF-ß-induced cellular processes, such as the cytostatic response, extracellular matrix remodeling, and epithelial dedifferentiation. In addition, we identified proteins involved in other cellular functions, such as vesicle trafficking, that were not previously associated with TGF-ß signaling. Although many TGF-ß responses are mediated by phosphorylation of the transcriptional regulators of the SMAD family by the TGF-ß receptor complex, we observed rapid kinetics of changes in protein phosphorylation, indicating that many responses were mediated through SMAD-independent TGF-ß signaling. Combined analysis of changes in protein abundance and phosphorylation and knowledge of protein interactions and transcriptional regulation provided a comprehensive representation of the dynamic signaling events underlying TGF-ß-induced changes in cell behavior. Our data suggest that in epithelial cells stimulated with TGF-ß, early signaling is a mixture of both pro- and antiproliferative signals, whereas later signaling primarily inhibits proliferation.


Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Queratinócitos/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Queratinócitos/citologia , Fatores de Tempo
14.
PLoS One ; 9(1): e86273, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24489709

RESUMO

ENDOGLIN (ENG) is a co-receptor for transforming growth factor-ß (TGF-ß) family members that is highly expressed in endothelial cells and has a critical function in the development of the vascular system. Mutations in Eng are associated with the vascular disease known as hereditary hemorrhagic telangiectasia type l. Using mouse embryonic stem cells we observed that angiogenic factors, including vascular endothelial growth factor (VEGF), induce vasculogenesis in embryoid bodies even when Eng deficient cells or cells depleted of Eng using shRNA are used. However, ENG is required for the stem cell-derived endothelial cells to organize effectively into tubular structures. Consistent with this finding, fetal metatarsals isolated from E17.5 Eng heterozygous mouse embryos showed reduced VEGF-induced vascular network formation. Moreover, shRNA-mediated depletion and pharmacological inhibition of ENG in human umbilical vein cells mitigated VEGF-induced angiogenesis. In summary, we demonstrate that ENG is required for efficient VEGF-induced angiogenesis.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Endoglina , Citometria de Fluxo , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Neovascularização Fisiológica/genética
15.
PLoS One ; 8(11): e81710, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24312339

RESUMO

The cystine-knot containing protein Sclerostin is an important negative regulator of bone growth and therefore represents a promising therapeutic target. It exerts its biological task by inhibiting the Wnt (wingless and int1) signaling pathway, which participates in bone formation by promoting the differentiation of mesenchymal stem cells to osteoblasts. The core structure of Sclerostin consists of three loops with the first and third loop (Finger 1 and Finger 2) forming a structured ß-sheet and the second loop being unstructured and highly flexible. Biochemical data showed that the flexible loop is important for binding of Sclerostin to Wnt co-receptors of the low-density lipoprotein related-protein family (LRP), by interacting with the Wnt co-receptors LRP5 or -6 it inhibits Wnt signaling. To further examine the structural requirements for Wnt inhibition, we performed an extensive mutational study within all three loops of the Sclerostin core domain involving single and multiple mutations as well as truncation of important regions. By this approach we could confirm the importance of the second loop and especially of amino acids Asn92 and Ile94 for binding to LRP6. Based on a Sclerostin variant found in a Turkish family suffering from Sclerosteosis we generated a Sclerostin mutant with cysteines 84 and 142 exchanged thereby removing the third disulfide bond of the cystine-knot. This mutant binds to LRP6 with reduced binding affinity and also exhibits a strongly reduced inhibitory activity against Wnt1 thereby showing that also elements outside the flexible loop are important for inhibition of Wnt by Sclerostin. Additionally, we examined the effect of the mutations on the inhibition of two different Wnt proteins, Wnt3a and Wnt1. We could detect clear differences in the inhibition of these proteins, suggesting that the mechanism by which Sclerostin antagonizes Wnt1 and Wnt3a is fundamentally different.


Assuntos
Cistina , Análise Mutacional de DNA , Glicoproteínas/química , Glicoproteínas/metabolismo , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Animais , Glicoproteínas/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Modelos Moleculares , Relação Estrutura-Atividade , Proteína Wnt1/antagonistas & inibidores , Proteína Wnt3A/antagonistas & inibidores
16.
PLoS One ; 8(4): e62295, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23638027

RESUMO

Sclerosteosis is a rare high bone mass disease that is caused by inactivating mutations in the SOST gene. Its gene product, Sclerostin, is a key negative regulator of bone formation and might therefore serve as a target for the anabolic treatment of osteoporosis. The exact molecular mechanism by which Sclerostin exerts its antagonistic effects on Wnt signaling in bone forming osteoblasts remains unclear. Here we show that Wnt3a-induced transcriptional responses and induction of alkaline phosphatase activity, an early marker of osteoblast differentiation, require the Wnt co-receptors LRP5 and LRP6. Unlike Dickkopf1 (DKK1), Sclerostin does not inhibit Wnt-3a-induced phosphorylation of LRP5 at serine 1503 or LRP6 at serine 1490. Affinity labeling of cell surface proteins with [(125)I]Sclerostin identified LRP6 as the main specific Sclerostin receptor in multiple mesenchymal cell lines. When cells were challenged with Sclerostin fused to recombinant green fluorescent protein (GFP) this was internalized, likely via a Clathrin-dependent process, and subsequently degraded in a temperature and proteasome-dependent manner. Ectopic expression of LRP6 greatly enhanced binding and cellular uptake of Sclerostin-GFP, which was reduced by the addition of an excess of non-GFP-fused Sclerostin. Finally, an anti-Sclerostin antibody inhibited the internalization of Sclerostin-GFP and binding of Sclerostin to LRP6. Moreover, this antibody attenuated the antagonistic activity of Sclerostin on canonical Wnt-induced responses.


Assuntos
Anticorpos Neutralizantes/imunologia , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Especificidade de Anticorpos , Diferenciação Celular , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Fosforilação , Transporte Proteico
17.
J Bone Miner Res ; 27(10): 2065-74, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22589174

RESUMO

The Wnt/ß-catenin signaling pathway plays a pivotal role in regulating osteoblast differentiation and bone formation. Here, we identify low-density lipoprotein (LDL) receptor-related protein 8 (LRP8) as a positive regulator of Wnt/ß-catenin signaling. In a small interfering RNA (siRNA) screen, LRP8 was shown to be required for Wnt/ß-catenin-induced transcriptional reporter activity. We found that ectopic expression of LRP8 increased Wnt-induced transcriptional responses, and promoted Wnt-induced ß-catenin accumulation. Moreover, knockdown of LRP8 resulted in a decrease in ß-catenin levels and suppression of Wnt/ß-catenin-induced Axin2 transcription. Functional studies in KS483 osteoprogenitor cells showed that LRP8 depletion resulted in impaired activation of endogenous Wnt-induced genes and decreased osteoblast differentiation and mineralization, whereas LRP8 ectopic expression had the opposite effect. These results identify LRP8 as a novel positive factor of canonical Wnt signaling pathway and show its involvement in Wnt-induced osteoblast differentiation.


Assuntos
Diferenciação Celular , Proteínas Relacionadas a Receptor de LDL/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Via de Sinalização Wnt , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo , Animais , Calcificação Fisiológica , Linhagem Celular , Núcleo Celular/metabolismo , Genes Reporter , Humanos , Proteínas Relacionadas a Receptor de LDL/deficiência , Proteínas Relacionadas a Receptor de LDL/genética , Camundongos , Estabilidade Proteica , Transcrição Genética
18.
Endocrinology ; 153(3): 1509-18, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22294741

RESUMO

Genetic studies have identified bone morphogenetic protein-15 (BMP15) as an essential regulator of female fertility in humans and in sheep. Oocyte-derived BMP15 is a noncovalently linked dimeric growth factor mediating its effects to ovarian somatic cells in a paracrine manner. Although receptor ectodomains capable of binding BMP15 have previously been reported, no cell surface receptor complex involved in BMP15 signaling has previously been characterized. Here we have expressed and purified recombinant human BMP15 noncovalent and covalent dimer variants. The biological effects of these BMP15 variants were assessed in cultured human granulosa-luteal cells or COV434 granulosa cell tumor cells using BMP-responsive transcriptional reporter assays and an inhibin B ELISA. Biochemical characterization of ligand-receptor interactions was performed with affinity-labeling experiments using [(125)I]iodinated BMP15 variants. Both ligand variants were shown to form homodimers and to stimulate Smad1/5/8 signaling and inhibin B production in human granulosa cells in a similar manner. [(125)I]Iodination of both ligands was achieved, but only the covalent dimer variant retained receptor binding capacity. The [(125)I]BMP15(S356C) variant bound preferentially to endogenous BMP receptor 1B (BMPR1B) and BMPR2 receptors on COV434 cells. Binding experiments in COS cells with overexpression of these receptors confirmed that the [(125)I]BMP15(S356C) variant binds to BMPR1B and BMPR2 forming the BMP15 signaling complex. The results provide the first direct evidence in any species on the identification of specific cell surface receptors for a member of the GDF9/BMP15 subfamily of oocyte growth factors. The fact that BMP15 uses preferentially BMPR1B as its type I receptor suggests an important role for the BMPR1B receptor in human female fertility. The result is well in line with the demonstration of ovarian failure in a recently reported human subject with a homozygous BMPR1B loss-of-function mutant.


Assuntos
Proteína Morfogenética Óssea 15/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Membrana Celular/metabolismo , Regulação da Expressão Gênica , Células da Granulosa/citologia , Ovário/metabolismo , Animais , Células COS , Chlorocebus aethiops , Dimerização , Feminino , Genes Reporter , Células da Granulosa/metabolismo , Homozigoto , Humanos , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química
19.
J Bone Miner Res ; 26(6): 1178-87, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21611961

RESUMO

Bone morphogenetic proteins (BMPs) exert an important role in skeletal development, adult bone homeostasis, and fracture healing and have demonstrated clinical utility for bone regeneration. However, BMPs fall short as regenerative agents because high doses need to be used to obtain therapeutic effects. Determining the molecular mechanisms controlling BMP-induced bone formation may lead to the development of more effective BMP-based therapies. To identify kinases mediating BMP-induced osteoblast differentiation, we performed an siRNA screen to find kinases modulating BMP-6-induced alkaline phosphatase (ALP) activity. Surprisingly, although transforming growth factor ß (TGF-ß) generally is considered to antagonize BMP-induced osteoblast differentiation, C2C12 cells transfected with siRNAs targeting TGF-ß receptors displayed reduced BMP-6-induced ALP activity. Furthermore, pharmacologic inhibitors blocking the TGF-ß type I receptor impaired BMP-induced ALP activity in KS483 and C2C12 cells and mineralization of KS483 cells. Consistently, costimulation with BMPs and TGF-ß further increased expression of osteoblast-specific genes, ALP activity, and mineralization of KS483 cells and primary mesenchymal stem cells compared with BMPs alone. The stimulatory and inhibitory effects of TGF-ß were found to depend on timing and duration of the costimulation. TGF-ß inhibited BMP-induced activation of a BMP-Smad-dependent luciferase reporter, suggesting that the stimulatory effect of TGF-ß is not due to increased BMP-Smad activity. TGF-ß also inhibited the BMP-induced expression of the BMP antagonist noggin and prolonged BMP activity. In conclusion, TGF-ß, besides acting as an inhibitor, also can, by dampening the noggin-mediated negative-feedback loop, enhance BMP-induced osteoblast differentiation, which might be beneficial in fracture healing.


Assuntos
Proteína Morfogenética Óssea 6/farmacologia , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Osteoblastos/enzimologia , RNA Interferente Pequeno/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
20.
Eur J Hum Genet ; 19(4): 389-93, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21248739

RESUMO

Down's syndrome (DS), resulting from an additional copy of chromosome 21 (trisomy 21), is frequently associated with congenital heart defects (CHDs). Although the increased dosage of chromosome 21 sequences is likely to be part of the etiology of cardiac defects, only a proportion of DS patients exhibit a congenital heart defect (birth prevalence 40-60%). Through a large-candidate gene-sequencing screen in patients with atrioventricular septal defects, substitutions were identified in bone morphogenetic protein (BMP) type I receptor ALK2 and two other genes in a patient with DS and a primum-type atrial septal defect. Structural modeling of the cytoplasmic domain of the ALK2 receptor suggests that H286 is in close proximity to the nucleotide-binding site of the kinase domain. We investigated whether this p.His286Asp substitution altered ALK2 function by using both in vitro as well as in vivo assays. The p.His286Asp variant demonstrated impaired functional activity as measured by BMP-specific transcriptional response assays. Furthermore, mild dominant-interfering activity was observed in vivo compared with wild-type ALK2 as determined by RNA injection into zebrafish embryos. These data indicate that in the context of a DS background, ALK2-mediated reduction of BMP signaling may contribute to CHDs.


Assuntos
Receptores de Ativinas Tipo I/genética , Síndrome de Down/genética , Cardiopatias Congênitas/etiologia , Cardiopatias Congênitas/genética , Mutação/genética , Receptores de Ativinas Tipo I/química , Receptores de Ativinas Tipo I/metabolismo , Animais , Proteína Morfogenética Óssea 1/metabolismo , Bovinos , Síndrome de Down/complicações , Feminino , Cardiopatias Congênitas/diagnóstico , Comunicação Interatrial/genética , Comunicação Interatrial/patologia , Humanos , Masculino , Conformação Proteica , Peixe-Zebra/genética
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