RESUMO
The bone morphogenetic protein (BMP) signaling pathway plays pivotal roles in various biological processes during embryogenesis and adult homeostasis. Transmembrane anterior posterior transformation 1 (TAPT1) is an evolutionarily conserved protein involved in murine axial skeletal patterning. Genetic defects in TAPT1 result in complex lethal osteochondrodysplasia. However, the specific cellular activity of TAPT1 is not clear. Herein, we report that TAPT1 inhibits BMP signaling and destabilizes the SMAD1/5 protein by facilitating its interaction with SMURF1 E3 ubiquitin ligase, which leads to SMAD1/5 proteasomal degradation. In addition, we found that the activation of BMP signaling facilitates the redistribution of TAPT1 and promotes its association with SMAD1. TAPT1-deficient murine C2C12 myoblasts or C3H/10T1/2 mesenchymal stem cells exhibit elevated SMAD1/5/9 protein levels, which amplifies BMP activation, in turn leading to a boost in the transdifferentiation or differentiation processing of these distinct TAPT1-deficient cell lines changing into mature osteoblasts. Furthermore, the enhancing effect of TAPT1 deficiency on osteogenic differentiation of C3H/10T1/2 cells was observed in an in vivo ectopic bone formation model. Importantly, a subset of TAPT1 mutations identified in humans with lethal skeletal dysplasia exhibited gain-of-function activity on SMAD1 protein levels. Thus, this finding elucidates the role of TAPT1 in the regulation of SMAD1/5 protein stability for controlling BMP signaling.
Assuntos
Transdução de Sinais , Proteína Smad1 , Proteína Smad5 , Animais , Humanos , Camundongos , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular , Linhagem Celular , Proteínas de Membrana , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , Estabilidade Proteica , Transdução de Sinais/genética , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Proteína Smad8/genética , Proteína Smad8/metabolismoRESUMO
RESEARCH QUESTION: What is the effect of miR-122 on the progression and recovery of fibrosis in Asherman's syndrome? DESIGN: Endometrial tissue was collected from 21 patients, 11 with intrauterine adhesion (IUA) and 10 without IUA. Quantitative real-time polymerase chain reaction, immunofluorescence and Western blot were applied to observe the expression of mRNAs/miRNAs and protein, respectively. The endometrial physical injury was carried out in C57BL/6 mice to create an endometrial fibrosis model, with intrauterine injection of adenovirus to compare the antifibrosis and repair function of miR-122 on endometrium. The morphology of the uterus was observed using haematoxylin and eosin staining, and fibrosis markers were detected by immunohistochemistry. RESULTS: miR-122 expression was reduced in patients with IUAs, accompanied by fibrosis. MiR-122 overexpression reduced the degree of fibrosis in endometrial stromal cells. Further molecular analyses demonstrated that miR-122 inhibited fibrosis through the TGF-ß/SMAD pathway by directly targeting the 3' untranslated region of SMAD family member 3, suppressing its expression. Notably, miR-122 promoted endometrial regeneration and recovery of pregnancy capacity in a mouse endometrial injury model. CONCLUSIONS: miR-122 is a critical regulator for repair of endometrial fibrosis and provided new insight for the clinical treatment of intrauterine adhesions.
Assuntos
Ginatresia , MicroRNAs , Doenças Uterinas , Camundongos , Animais , Feminino , Gravidez , Humanos , Fator de Crescimento Transformador beta/metabolismo , Camundongos Endogâmicos C57BL , Doenças Uterinas/genética , Doenças Uterinas/patologia , Endométrio/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Aderências Teciduais , Modelos Animais de Doenças , FibroseRESUMO
Understanding signaling pathways that regulate pancreatic ß-cell function to produce, store, and release insulin, as well as pathways that control ß-cell proliferation, is vital to find new treatments for diabetes mellitus. Transforming growth factor-beta (TGF-ß) signaling is involved in a broad range of ß-cell functions. The canonical TGF-ß signaling pathway functions through intracellular smads, including smad2 and smad3, to regulate cell development, proliferation, differentiation, and function in many organs. Here, we demonstrate the role of TGF-ß/smad2 signaling in regulating mature ß-cell proliferation and function using ß-cell-specific smad2 null mutant mice. ß-cell-specific smad2-deficient mice exhibited improved glucose clearance as demonstrated by glucose tolerance testing, enhanced in vivo and ex vivo glucose-stimulated insulin secretion, and increased ß-cell mass and proliferation. Furthermore, when these mice were fed a high-fat diet to induce hyperglycemia, they again showed improved glucose tolerance, insulin secretion, and insulin sensitivity. In addition, ex vivo analysis of smad2-deficient islets showed that they displayed increased glucose-stimulated insulin secretion and upregulation of genes involved in insulin synthesis and insulin secretion. Thus, we conclude that smad2 could represent an attractive therapeutic target for type 2 diabetes mellitus.
Assuntos
Hiperglicemia/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Hiperglicemia/induzido quimicamente , Hiperglicemia/genética , Camundongos , Camundongos Knockout , Proteína Smad2/genéticaRESUMO
Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are co-expressed exclusively in oocytes throughout most of folliculogenesis and play central roles in controlling ovarian physiology. Although both growth factors exist as homodimers, recent evidence indicates that GDF9 and BMP15 can also heterodimerize to form the potent growth factor cumulin. Within the cumulin complex, BMP15 "activates" latent GDF9, enabling potent signaling in granulosa cells via type I receptors (i.e. activin receptor-like kinase-4/5 (ALK4/5)) and SMAD2/3 transcription factors. In the cumulin heterodimer, two distinct type I receptor interfaces are formed compared with homodimeric GDF9 and BMP15. Previous studies have highlighted the potential of cumulin to improve treatment of female infertility, but, as a noncovalent heterodimer, cumulin is difficult to produce and purify without contaminating GDF9 and BMP15 homodimers. In this study we addressed this challenge by focusing on the cumulin interface formed by the helix of the GDF9 chain and the fingers of the BMP15 chain. We demonstrate that unique BMP15 finger residues at this site (Arg301, Gly304, His307, and Met369) enable potent activation of the SMAD2/3 pathway. Incorporating these BMP15 residues into latent GDF9 generated a highly potent growth factor, called hereafter Super-GDF9. Super-GDF9 was >1000-fold more potent than WT human GDF9 and 4-fold more potent than cumulin in SMAD2/3-responsive transcriptional assays in granulosa cells. Our demonstration that Super-GDF9 can effectively promote mouse cumulus cell expansion and improve oocyte quality in vitro represents a potential solution to the current challenges of producing and purifying intact cumulin.
Assuntos
Fator 9 de Diferenciação de Crescimento/metabolismo , Oócitos/metabolismo , Animais , Proteína Morfogenética Óssea 15/genética , Proteína Morfogenética Óssea 15/metabolismo , Linhagem Celular Tumoral , Feminino , Variação Genética/genética , Fator 9 de Diferenciação de Crescimento/genética , Humanos , Camundongos , Modelos Moleculares , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismoRESUMO
The interplay between the transforming growth factor ß (TGF-ß) signaling proteins, SMAD family member 2 (SMAD2) and 3 (SMAD3), and the TGF-ß-inhibiting SMAD, SMAD7, seems to play a vital role in proper pancreatic endocrine development and also in normal ß-cell function in adult pancreatic islets. Here, we generated conditional SMAD7 knockout mice by crossing insulin1Cre mice with SMAD7fx/fx mice. We also created a ß cell-specific SMAD7-overexpressing mouse line by crossing insulin1Dre mice with HPRT-SMAD7/RosaGFP mice. We analyzed ß-cell function in adult islets when SMAD7 was either absent or overexpressed in ß cells. Loss of SMAD7 in ß cells inhibited proliferation, and SMAD7 overexpression enhanced cell proliferation. However, alterations in basic glucose homeostasis were not detectable following either SMAD7 deletion or overexpression in ß cells. Our results show that both the absence and overexpression of SMAD7 affect TGF-ß signaling and modulates ß-cell proliferation but does not appear to alter ß-cell function. Reversible SMAD7 overexpression may represent an attractive therapeutic option to enhance ß-cell proliferation without negative effects on ß-cell function.
Assuntos
Proliferação de Células , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Insulina/fisiologia , Proteína Smad7/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Feminino , Glucose/farmacologia , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais , Edulcorantes/farmacologia , Fator de Crescimento Transformador beta/genéticaRESUMO
Caloric restriction has been associated with increased life span and reduced aging-related disorders and reduces fibrosis in several diseases. Fibrosis is characterized by deposition of excess fibrous material in tissues and organs and is caused by aging, chronic stress, injury, or disease. Myofibroblasts are fibroblast-like cells that secrete high levels of extracellular matrix proteins, resulting in fibrosis. Histological studies have identified many-fold increases of myofibroblasts in aged organs where myofibroblasts are constantly generated from resident tissue fibroblasts and other cell types. However, it remains unclear how aging increases the generation of myofibroblasts. Here, using mouse models and biochemical assays, we show that sirtuin 6 (SIRT6) deficiency plays a major role in aging-associated transformation of fibroblasts to myofibroblasts, resulting in tissue fibrosis. Our findings suggest that SIRT6-deficient fibroblasts transform spontaneously to myofibroblasts through hyperactivation of transforming growth factor ß (TGF-ß) signaling in a cell-autonomous manner. Importantly, we noted that SIRT6 haploinsufficiency is sufficient for enhancing myofibroblast generation, leading to multiorgan fibrosis and cardiac dysfunction in mice during aging. Mechanistically, SIRT6 bound to and repressed the expression of key TGF-ß signaling genes by deacetylating SMAD family member 3 (SMAD3) and Lys-9 and Lys-56 in histone 3. SIRT6 binding to the promoters of genes in the TGF-ß signaling pathway decreased significantly with age and was accompanied by increased binding of SMAD3 to these promoters. Our findings reveal that SIRT6 may be a potential candidate for modulating TGF-ß signaling to reduce multiorgan fibrosis during aging and fibrosis-associated diseases.
Assuntos
Fibroblastos/patologia , Miocárdio/patologia , Sirtuínas/genética , Fator de Crescimento Transformador beta/genética , Envelhecimento , Animais , Fibroblastos/metabolismo , Fibrose , Deleção de Genes , Masculino , Camundongos , Miocárdio/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Transdução de Sinais , Proteína Smad3/metabolismo , Ativação Transcricional , Fator de Crescimento Transformador beta/metabolismoRESUMO
Modification of the transforming growth factor ß (TGF-ß) signaling components by (de)ubiquitination is emerging as a key regulatory mechanism that controls cell signaling responses in health and disease. Here, we show that the deubiquitinating enzyme UBH-1 in Caenorhabditis elegans and its human homolog, ubiquitin C-terminal hydrolase-L1 (UCH-L1), stimulate DAF-7/TGF-ß signaling, suggesting that this mode of regulation of TGF-ß signaling is conserved across animal species. The dauer larva-constitutive C. elegans phenotype caused by defective DAF-7/TGF-ß signaling was enhanced and suppressed, respectively, by ubh-1 deletion and overexpression in the loss-of-function genetic backgrounds of daf7, daf-1/TGF-ßRI, and daf4/R-SMAD, but not of daf-8/R-SMAD. This suggested that UBH-1 may stimulate DAF-7/TGF-ß signaling via DAF-8/R-SMAD. Therefore, we investigated the effect of UCH-L1 on TGF-ß signaling via its intracellular effectors, i.e. SMAD2 and SMAD3, in mammalian cells. Overexpression of UCH-L1, but not of UCH-L3 (the other human homolog of UBH1) or of the catalytic mutant UCH-L1C90A, enhanced TGF-ß/SMAD-induced transcriptional activity, indicating that the deubiquitination activity of UCH-L1 is indispensable for enhancing TGF-ß/SMAD signaling. We also found that UCH-L1 interacts, deubiquitinates, and stabilizes SMAD2 and SMAD3. Under hypoxia, UCH-L1 expression increased and TGF-ß/SMAD signaling was potentiated in the A549 human lung adenocarcinoma cell line. Notably, UCH-L1-deficient A549 cells were impaired in tumorigenesis, and, unlike WT UCH-L1, a UCH-L1 variant lacking deubiquitinating activity was unable to restore tumorigenesis in these cells. These results indicate that UCH-L1 activity supports DAF-7/TGF-ß signaling and suggest that UCH-L1's deubiquitination activity is a potential therapeutic target for managing lung cancer.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Carcinogênese/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Caenorhabditis elegans , Transformação Celular Neoplásica , Enzimas Desubiquitinantes , Larva/metabolismo , Pulmão/metabolismo , Transdução de Sinais/genética , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Ubiquitina Tiolesterase/fisiologia , UbiquitinaçãoRESUMO
Cytochrome P450 1A1 (CYP1A1) catalyzes the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) and is transcriptionally regulated by the aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) complex upon exposure to PAHs. Accordingly, inhibition of CYP1A1 expression reduces production of carcinogens from PAHs. Although transcription of the CYP1A1 gene is known to be repressed by transforming growth factor-ß (TGF-ß), how TGF-ß signaling is involved in the suppression of CYP1A1 gene expression has yet to be clarified. In this study, using mammalian cell lines, along with shRNA-mediated gene silencing, CRISPR/Cas9-based genome editing, and reporter gene and quantitative RT-PCR assays, we found that TGF-ß signaling dissociates the B[a]P-mediated AhR/ARNT heteromeric complex. Among the examined Smads, Smad family member 3 (Smad3) strongly interacted with both AhR and ARNT via its MH2 domain. Moreover, hypoxia-inducible factor 1α (HIF-1α), which is stabilized upon TGF-ß stimulation, also inhibited AhR/ARNT complex formation in the presence of B[a]P. Thus, TGF-ß signaling negatively regulated the transcription of the CYP1A1 gene in at least two different ways. Of note, TGF-ß abrogated DNA damage in B[a]P-exposed cells. We therefore conclude that TGF-ß may protect cells against carcinogenesis because it inhibits CYP1A1-mediated metabolic activation of PAHs as part of its anti-tumorigenic activities.
Assuntos
Citocromo P-450 CYP1A1/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células A549 , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Benzo(a)pireno/toxicidade , Células COS , Chlorocebus aethiops , Citocromo P-450 CYP1A1/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Pirenos , Transdução de Sinais , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Mediator complex subunit 16 (MED16) is a component of the mediator complex and functions as a coactivator in transcriptional events at almost all RNA polymerase II-dependent genes. In this study, we report that the expression of MED16 is markedly decreased in papillary thyroid cancer (PTC) tumors compared with normal thyroid tissues. In vitro, MED16 overexpression in PTC cells significantly inhibited cell migration, enhanced sodium/iodide symporter expression and iodine uptake, and decreased resistance to radioactive 131I (RAI). Conversely, PTC cells in which MED16 had been further knocked down (MED16KD) exhibited enhanced cell migration, epithelial-mesenchymal transition, and RAI resistance, accompanied by decreased sodium/iodide symporter levels. Moreover, cell signaling through transforming growth factor ß (TGF-ß) was highly activated after the MED16 knockdown. Similar results were obtained in MED12KD PTC cells, and a co-immunoprecipitation experiment verified interactions between MED16 and MED12 and between MED16 and TGF-ßR2. Of note, the application of LY2157299, a potent inhibitor of TGF-ß signaling, significantly attenuated MED16KD-induced RAI resistance both in vitro and in vivo In conclusion, our findings indicate that MED16 reduction in PTC contributes to tumor progression and RAI resistance via the activation of the TGF-ß pathway.
Assuntos
Radioisótopos do Iodo/farmacologia , Complexo Mediador/metabolismo , Proteínas de Neoplasias/metabolismo , Tolerância a Radiação , Transdução de Sinais , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Complexo Mediador/genética , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Câncer Papilífero da Tireoide/metabolismo , Câncer Papilífero da Tireoide/patologia , Câncer Papilífero da Tireoide/radioterapia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/radioterapiaRESUMO
Hypoxia-inducible factor 2α (HIF2α) directly regulates a battery of genes essential for intestinal iron absorption. Interestingly, iron deficiency and overload disorders do not result in increased intestinal expression of glycolytic or angiogenic HIF2α target genes. Similarly, inflammatory and tumor foci can induce a distinct subset of HIF2α target genes in vivo These observations indicate that different stimuli activate distinct subsets of HIF2α target genes via mechanisms that remain unclear. Here, we conducted a high-throughput siRNA-based screen to identify genes that regulate HIF2α's transcriptional activity on the promoter of the iron transporter gene divalent metal transporter-1 (DMT1). SMAD family member 3 (SMAD3) and SMAD4 were identified as potential transcriptional repressors. Further analysis revealed that SMAD4 signaling selectively represses iron-absorptive gene promoters but not the inflammatory or glycolytic HIF2α or HIF1α target genes. Moreover, the highly homologous SMAD2 did not alter HIF2α transcriptional activity. During iron deficiency, SMAD3 and SMAD4 expression was significantly decreased via proteasomal degradation, allowing for derepression of iron target genes. Several iron-regulatory genes contain a SMAD-binding element (SBE) in their proximal promoters; however, mutation of the putative SBE on the DMT1 promoter did not alter the repressive function of SMAD3 or SMAD4. Importantly, the transcription factor forkhead box protein A1 (FOXA1) was critical in SMAD4-induced DMT1 repression, and DNA binding of SMAD4 was essential for the repression of HIF2α activity, suggesting an indirect repressive mechanism through DNA binding. These results provide mechanistic clues to how HIF signaling can be regulated by different cellular cues.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Reguladoras de Ferro/metabolismo , Proteína Smad3/metabolismo , Proteína Smad4/metabolismo , Animais , Células Cultivadas , Humanos , Proteínas Reguladoras de Ferro/genética , Camundongos , Camundongos Knockout , Proteína Smad3/deficiência , Proteína Smad4/deficiênciaRESUMO
Bone morphogenetic protein 10 (BMP10) is a cardiac peptide growth factor belonging to the transforming growth factor ß superfamily that critically controls cardiovascular development, growth, and maturation. It has been shown that BMP10 elicits its intracellular signaling through a receptor complex of activin receptor-like kinase 1 with morphogenetic protein receptor type II or activin receptor type 2A. Previously, we generated and characterized a transgenic mouse line expressing BMP10 from the α-myosin heavy chain gene promoter and found that these mice have normal cardiac hypertrophic responses to both physiological and pathological stimuli. In this study, we report that these transgenic mice exhibit significantly reduced levels of cardiomyocyte apoptosis and cardiac fibrosis in response to a prolonged administration of the ß-adrenoreceptor agonist isoproterenol. We further confirmed this cardioprotective function with a newly generated conditional Bmp10 transgenic mouse line, in which Bmp10 was activated in adult hearts by tamoxifen. Moreover, the intraperitoneal administration of recombinant human BMP10 was found to effectively protect hearts from injury, suggesting potential therapeutic utility of using BMP10 to prevent heart failure. Gene profiling and biochemical analyses indicated that BMP10 activates the SMAD-mediated canonical pathway and, unexpectedly, also the signal transducer and activator of transcription 3 (STAT3)-mediated signaling pathway both in vivo and in vitro Additional findings further supported the notion that BMP10's cardioprotective function likely is due to its dual activation of SMAD- and STAT3-regulated signaling pathways, promoting cardiomyocyte survival and suppressing cardiac fibrosis.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Smad/metabolismo , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/genética , Matriz Extracelular/metabolismo , Coração/efeitos dos fármacos , Humanos , Isoproterenol/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Functional activation of the transforming growth factor-ß (TGF-ß) receptors (TGFBRs) is carefully regulated through integration of post-translational modifications, spatial regulation at the cellular level, and TGFBR availability at the cell surface. Although the bulk of TGFBRs resides inside the cells, AKT Ser/Thr kinase (AKT) activation in response to insulin or other growth factors rapidly induces transport of TGFBRs to the cell surface, thereby increasing the cell's responsiveness to TGF-ß. We now demonstrate that TGF-ß itself induces a rapid translocation of its own receptors to the cell surface and thus amplifies its own response. This mechanism of response amplification, which hitherto has not been reported for other cell-surface receptors, depended on AKT activation and TGF-ß type I receptor kinase. In addition to an increase in cell-surface TGFBR levels, TGF-ß treatment promoted TGFBR internalization, suggesting an overall amplification of TGFBR cycling. The TGF-ß-induced increase in receptor presentation at the cell surface amplified TGF-ß-induced SMAD family member (SMAD) activation and gene expression. Furthermore, bone morphogenetic protein 4 (BMP-4), which also induces AKT activation, increased TGFBR levels at the cell surface, leading to enhanced autocrine activation of TGF-ß-responsive SMADs and gene expression, providing context for the activation of TGF-ß signaling in response to BMP during development. In summary, our results indicate that TGF-ß- and BMP-induced activation of low levels of cell surface-associated TGFBRs rapidly mobilizes additional TGFBRs from intracellular stores to the cell surface, increasing the abundance of cell-surface TGFBRs and cells' responsiveness to TGF-ß signaling.
Assuntos
Receptor do Fator de Crescimento Transformador beta Tipo I/biossíntese , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Células A549 , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Humanos , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Proteínas Smad/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
Smad proteins are transcriptional regulators activated by TGF-ß. They are known to bind to two distinct Smad-responsive motifs, namely the Smad-binding element (SBE) (5'-GTCTAGAC-3') and CAGA motifs (5'-AGCCAGACA-3' or 5'-TGTCTGGCT-3'). However, the mechanisms by which these motifs promote Smad activity are not fully elucidated. In this study, we performed DNA CASTing, binding assays, ChIP sequencing, and quantitative RT-PCR to dissect the details of Smad binding and function of the SBE and CAGA motifs. We observed a preference for Smad3 to bind CAGA motifs and Smad4 to bind SBE, and that either one SBE or a triple-CAGA motif forms a cis-acting functional half-unit for Smad-dependent transcription activation; combining two half-units allows efficient activation. Unexpectedly, the extent of Smad binding did not directly correlate with the abilities of Smad-binding sequences to induce gene expression. We found that Smad proteins are more tolerant of single bp mutations in the context of the CAGA motifs, with any mutation in the SBE disrupting function. CAGA and CAGA-like motifs but not SBE are widely distributed among stimulus-dependent Smad2/3-binding sites in normal murine mammary gland epithelial cells, and the number of CAGA and CAGA-like motifs correlates with fold-induction of target gene expression by TGF-ß. These data, demonstrating Smad responsiveness can be tuned by both sequence and number of repeats, provide a compelling explanation for why CAGA motifs are predominantly used for Smad-dependent transcription activation in vivo.
Assuntos
Proteína Smad3/química , Proteína Smad3/metabolismo , Proteína Smad4/química , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Motivos de Aminoácidos , Sequência de Bases , Sítios de Ligação , Humanos , Ligação Proteica , Elementos de Resposta , Proteína Smad2/química , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad4/genética , Ativação TranscricionalRESUMO
Bone morphogenetic protein (BMP) signaling is critical in renal development and disease. In animal models of chronic kidney disease (CKD), re-activation of BMP signaling is reported to be protective by promoting renal repair and regeneration. Clinical use of recombinant BMPs, however, requires harmful doses to achieve efficacy and is costly because of BMPs' complex synthesis. Therefore, alternative strategies are needed to harness the beneficial effects of BMP signaling in CKD. Key aspects of the BMP signaling pathway can be regulated by both extracellular and intracellular molecules. In particular, secreted proteins like noggin and chordin inhibit BMP activity, whereas kielin/chordin-like proteins (KCP) enhance it and attenuate kidney fibrosis or CKD. Clinical development of KCP, however, is precluded by its size and complexity. Therefore, we propose an alternative strategy to enhance BMP signaling by using small molecules, which are simpler to synthesize and more cost-effective. To address our objective, here we developed a small-molecule high-throughput screen (HTS) with human renal cells having an integrated luciferase construct highly responsive to BMPs. We demonstrate the activity of a potent benzoxazole compound, sb4, that rapidly stimulated BMP signaling in these cells. Activation of BMP signaling by sb4 increased the phosphorylation of key second messengers (SMAD-1/5/9) and also increased expression of direct target genes (inhibitors of DNA binding, Id1 and Id3) in canonical BMP signaling. Our results underscore the feasibility of utilizing HTS to identify compounds that mimic key downstream events of BMP signaling in renal cells and have yielded a lead BMP agonist.
Assuntos
Benzoxazóis/farmacologia , Proteínas Morfogenéticas Ósseas/agonistas , Proteínas Morfogenéticas Ósseas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Fosfoproteínas/metabolismo , Proteínas Smad/metabolismoRESUMO
TGFß signaling via SMAD proteins and protein kinase pathways up- or down-regulates the expression of many genes and thus affects physiological processes, such as differentiation, migration, cell cycle arrest, and apoptosis, during developmental or adult tissue homeostasis. We here report that NUAK family kinase 1 (NUAK1) and NUAK2 are two TGFß target genes. NUAK1/2 belong to the AMP-activated protein kinase (AMPK) family, whose members control central and protein metabolism, polarity, and overall cellular homeostasis. We found that TGFß-mediated transcriptional induction of NUAK1 and NUAK2 requires SMAD family members 2, 3, and 4 (SMAD2/3/4) and mitogen-activated protein kinase (MAPK) activities, which provided immediate and early signals for the transient expression of these two kinases. Genomic mapping identified an enhancer element within the first intron of the NUAK2 gene that can recruit SMAD proteins, which, when cloned, could confer induction by TGFß. Furthermore, NUAK2 formed protein complexes with SMAD3 and the TGFß type I receptor. Functionally, NUAK1 suppressed and NUAK2 induced TGFß signaling. This was evident during TGFß-induced epithelial cytostasis, mesenchymal differentiation, and myofibroblast contractility, in which NUAK1 or NUAK2 silencing enhanced or inhibited these responses, respectively. In conclusion, we have identified a bifurcating loop during TGFß signaling, whereby transcriptional induction of NUAK1 serves as a negative checkpoint and NUAK2 induction positively contributes to signaling and terminal differentiation responses to TGFß activity.
Assuntos
Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/metabolismoRESUMO
Bone morphogenetic proteins (BMPs) are important mediators of osteoclast differentiation. Although accumulating evidence has implicated BMPs in osteoblastogenesis, the mechanisms by which BMPs regulate osteoclastogenesis remain unclear. Activin A receptor type 1 (ACVR1) is a BMP type 1 receptor essential for skeletal development. Here, we observed that BMP-7, which preferentially binds to ACVR1, promotes osteoclast differentiation, suggesting ACVR1 is involved in osteoclastogenesis. To investigate this further, we isolated osteoclasts from either Acvr1-floxed mice or mice with constitutively-activated Acvr1 (caAcvr1) carrying tamoxifen-inducible Cre driven by a ubiquitin promotor and induced Cre activity in culture. Osteoclasts from the Acvr1-floxed mice had reduced osteoclast numbers and demineralization activity, whereas those from the caAcvr1-mutant mice formed large osteoclasts and demineralized pits, suggesting that BMP signaling through ACVR1 regulates osteoclast fusion and activity. It is reported that BMP-2 binds to BMPR1A, another BMP type 1 receptor, whereas BMP-7 binds to ACVR1 to activate SMAD1/5/9 signaling. Here, Bmpr1a-disrupted osteoclasts displayed reduced phospho-SMAD1/5/9 (pSMAD1/5/9) levels when induced by BMP-2, whereas no impacts on pSMAD1/5/9 were observed when induced by BMP-7. In contract, Acvr1-disrupted osteoclasts displayed reduced pSMAD1/5/9 levels when induced either by BMP-2 or BMP-7, suggesting that ACVR1 is the major receptor for transducing BMP-7 signals in osteoclasts. Indeed, LDN-193189 and LDN-212854, which specifically block SMAD1/5/9 phosphorylation, inhibited osteoclastogenesis of caAcvr1-mutant cells. Moreover, increased BMP signaling promoted nuclear translocation of nuclear factor-activated T-cells 1 (NFATc1), which was inhibited by LDN treatments. Taken together, ACVR1-mediated BMP-SMAD signaling activates NFATc1, a regulatory protein crucial for receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Osteogênese , Ligante RANK/farmacologia , Transdução de Sinais , Proteínas Smad/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Animais , Calcineurina/metabolismo , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Mutação/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologiaRESUMO
The forkhead box O (FOXO) proteins are transcription factors involved in the differentiation of many cell types. Type II collagen (Col2) Cre-Foxo1-knockout and Col2-Cre-Foxo1,3,4 triple-knockout mice exhibit growth plate malformation. Moreover, recent studies have reported that in some cells, the expressions and activities of FOXOs are promoted by transforming growth factor ß1 (TGFß1), a growth factor playing a key role in chondrogenic differentiation. Here, using a murine chondrogenic cell line (ATDC5), mouse embryos, and human mesenchymal stem cells, we report the mechanisms by which FOXOs affect chondrogenic differentiation. FOXO1 expression increased along with chondrogenic differentiation, and FOXO1 inhibition suppressed chondrogenic differentiation. TGFß1/SMAD signaling promoted expression and activity of FOXO1. In ATDC5, FOXO1 knockdown suppressed expression of sex-determining region Y box 9 (Sox9), a master regulator of chondrogenic differentiation, resulting in decreased collagen type II α1 (Col2a1) and aggrecan (Acan) expression after TGFß1 treatment. On the other hand, chemical FOXO1 inhibition suppressed Col2a1 and Acan expression without suppressing Sox9 To investigate the effects of FOXO1 on chondrogenic differentiation independently of SOX9, we examined FOXO1's effects on the cell cycle. FOXO1 inhibition suppressed expression of p21 and cell-cycle arrest in G0/G1 phase. Conversely, FOXO1 overexpression promoted expression of p21 and cell-cycle arrest. FOXO1 inhibition suppressed expression of nascent p21 RNA by TGFß1, and FOXO1 bound the p21 promoter. p21 inhibition suppressed expression of Col2a1 and Acan during chondrogenic differentiation. These results suggest that FOXO1 is necessary for not only SOX9 expression, but also cell-cycle arrest during chondrogenic differentiation via TGFß1 signaling.
Assuntos
Condrogênese/genética , Proteína Forkhead Box O1/genética , Fatores de Transcrição SOX9/genética , Fator de Crescimento Transformador beta1/genética , Agrecanas/genética , Animais , Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/genética , Colágeno Tipo II/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteína Forkhead Box O1/antagonistas & inibidores , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteínas Smad/genética , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Transforming growth factor (TGF)-ß signaling in humans is stringently regulated to prevent excessive TGF-ß signaling. In tumors, TGF-ß signaling can both negatively and positively regulate tumorigenesis dependent on tumor type, but the reason for these opposite effects is unclear. TGF-ß signaling is mainly mediated via the Smad-dependent pathway, and herein we found that PDZK1-interacting protein 1 (PDZK1IP1) interacts with Smad4. PDZK1IP1 inhibited both the TGF-ß and the bone morphogenetic protein (BMP) pathways without affecting receptor-regulated Smad (R-Smad) phosphorylation. Rather than targeting R-Smad phosphorylation, PDZK1IP1 could interfere with TGF-ß- and BMP-induced R-Smad/Smad4 complex formation. Of note, PDZK1IP1 retained Smad4 in the cytoplasm of TGF-ß-stimulated cells. To pinpoint PDZK1IP1's functional domain, we created several PDZK1IP1 variants and found that its middle region, from Phe40 to Ala49, plays a key role in its Smad4-regulating activity. PDZK1IP1 knockdown enhanced the expression of the TGF-ß target genes Smad7 and prostate transmembrane protein androgen-induced (TMEPAI) upon TGF-ß stimulation. In contrast, PDZK1IP1 overexpression suppressed TGF-ß-induced reporter activities, cell migration, and cell growth inhibition. In a xenograft tumor model in which TGF-ß was previously shown to elicit tumor-promoting effects, PDZK1IP1 gain of function decreased tumor size and increased survival rates. Taken together, these findings indicate that PDZK1IP1 interacts with Smad4 and thereby suppresses the TGF-ß signaling pathway.
Assuntos
Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Mapas de Interação de Proteínas , Transdução de Sinais , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Masculino , Camundongos Endogâmicos BALB C , FosforilaçãoRESUMO
Chondrocyte hypertrophy is the terminal step in chondrocyte differentiation and is crucial for endochondral bone formation. How signaling pathways regulate chondrocyte hypertrophic differentiation remains incompletely understood. In this study, using a Tbx18:Cre (Tbx18Cre/+) gene-deletion approach, we selectively deleted the gene for the signaling protein SMAD family member 4 (Smad4f/f ) in the limbs of mice. We found that the Smad4-deficient mice develop a prominent shortened limb, with decreased expression of chondrocyte differentiation markers, including Col2a1 and Acan, in the humerus at mid-to-late gestation. The most striking defects in these mice were the absence of stylopod elements and failure of chondrocyte hypertrophy in the humerus. Moreover, expression levels of the chondrocyte hypertrophy-related markers Col10a1 and Panx3 were significantly decreased. Of note, we also observed that the expression of runt-related transcription factor 2 (Runx2), a critical mediator of chondrocyte hypertrophy, was also down-regulated in Smad4-deficient limbs. To determine how the skeletal defects arose in the mouse mutants, we performed RNA-Seq with ChIP-Seq analyses and found that Smad4 directly binds to regulatory elements in the Runx2 promoter. Our results suggest a new mechanism whereby Smad4 controls chondrocyte hypertrophy by up-regulating Runx2 expression during skeletal development. The regulatory mechanism involving Smad4-mediated Runx2 activation uncovered here provides critical insights into bone development and pathogenesis of chondrodysplasia.
Assuntos
Desenvolvimento Ósseo , Condrócitos/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteína Smad4/genética , Animais , Diferenciação Celular , Proliferação de Células , Condrócitos/citologia , Condrócitos/metabolismo , Condrogênese , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Hipertrofia/genética , Hipertrofia/metabolismo , Hipertrofia/patologia , Camundongos , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Proteína Smad4/metabolismoRESUMO
Activin and Wnt signaling are necessary and sufficient for mesendoderm (ME) differentiation of human embryonic stem cells (ESCs). In this study, we report that during ME differentiation induced by Activin and Wnt, Activin/Smad2 induces a decrease of the repressive histone modification of H3K27me3 by promoting the proteasome-dependent degradation of enhancer of zeste 2 polycomb (EZH2)-repressive complex 2 subunit. As a result, recruitment of the forkhead protein FOXH1 on open chromatin regions integrates the signals of Activin/Smad2 and Wnt/ß-catenin to activate the expression of the ME genes including HAS2 and ALDH3A2 Consistently, H3K27me3 decrease is enriched on open chromatin around regulatory regions. Furthermore, knockdown of HAS2 or ALDH3A2 greatly attenuates ME differentiation. These findings unveil a pathway from extracellular signals to epigenetic modification-mediated gene activation during ME commitment.