RESUMO
BACKGROUND We studied the expression and effect of miR-26a-5p in synovial fibroblast in patients with rheumatoid arthritis (RA). MATERIAL AND METHODS The synovial tissues of 55 RA patients with total knee arthroplasty performed from January 2016 to December 2016 were collected as the RA group, and 62 patients without RA history amputation or total knee arthroplasty served as the control group. The expressions of miR-26a-5p and Smad 1 mRNA in synovial fibroblast in patients with RA were detected by qPCR; The expression of Smad 1 and TGF-ß1 protein in synovial tissue or synovial fibroblasts was detected by immunoblotting. Transwell assay was used to detect the invasive ability of synovial fibroblasts. RESULTS The expression of miR-26a-5p and Smad 1 in synovial fibroblast in patients with RA were significantly higher than those in the control group (P<0.05). The expression of miR-26a-5p in synovial tissue of RA patients was positively correlated with the expression of Smad 1 mRNA (r=0.8982, P<0.001). The luciferase system showed that miR-26a-5p targeting synovial membrane FLS cells (P<0.05); the expression of MMP-1, MMP-3, MMP-13, and TGF-b1 protein and mRNA in the synovial FLS cells of RA patients was significantly decreased; and the expression of miR-26a-5p was significantly decreased in FLS cells with invasive ability. CONCLUSIONS miR-26a-5p is highly expressed in synovial tissue of patients with RA, and its high expression can improve the invasive ability of synovial fibroblasts by targeting Smad 1 gene and accelerating the progression of RA.
Assuntos
Artrite Reumatoide/genética , MicroRNAs/biossíntese , Proteína Smad1/metabolismo , Membrana Sinovial/metabolismo , Adulto , Idoso , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Progressão da Doença , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Masculino , Metaloproteinases da Matriz/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Proteína Smad1/biossíntese , Proteína Smad1/genética , Membrana Sinovial/patologia , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genéticaRESUMO
The SMAD proteins are responsible for transducing signals from activated transforming growth factor-beta. This is the first study assessing the expression of SMAD-1/8, SMAD-2/3, SMAD-4, and SMAD-7 in chronic lymphocytic leukemia cells with regard to their clinical significance and potential prognostic value. Overexpression of SMAD-1/8 was observed in 160 chronic lymphocytic leukemia patients compared to 42 healthy volunteers (p = 0.023) and was associated with a more progressive course of the disease (p = 0.016). Moreover, the high expression of SMAD-1/8 correlated with other, well-established prognostic factors, including clinical stage (p = 0.010) and lymphocyte doubling time (p = 0.021). The expression of SMAD-4 was lower in chronic lymphocytic leukemia patients compared with the control group (p = 0.003). Importantly, lower SMAD-4 levels correlated with longer progression-free survival (p = 0.009), progressive course of the disease (p = 0.002), advanced clinical stage (p = 0.0004), elevated beta-2-microglobulin and lactate dehydrogenase levels (p < 0.05), shorter lymphocyte doubling time (p = 0.009), and CD38 antigen expression (p = 0.039). In addition, lower SMAD-4 expression correlated with lower apoptotic index (p = 0.0007) and lower expression of receptors for vascular endothelial growth factors VEGFR-1 and VEGFR-2. A significant association was found between the low expression of inhibitory protein SMAD-7 and both zeta-chain-associated protein kinase 70-negative cells (p = 0.04) and lower apoptotic index (p = 0.004). No differences were observed in SMAD-2/3 expression. In conclusion, our results demonstrate a significant correlation between greater SMAD-1/8 and lower SMAD-4 expression in chronic lymphocytic leukemia cells, as well as more progressive outcome and poor prognosis. These data provide supporting evidence that the expression of SMAD proteins plays an important role in disease development and may be considered as a novel, biologic prognostic factor in this disease.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Proteína Smad1/biossíntese , Proteína Smad2/biossíntese , Proteína Smad4/biossíntese , Proteína Smad7/biossíntese , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Intervalo Livre de Doença , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteína Smad1/genética , Proteína Smad2/genética , Proteína Smad4/genética , Proteína Smad7/genética , Fator de Crescimento Transformador beta/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Proteína-Tirosina Quinase ZAP-70/biossínteseRESUMO
Phosphodiesterase (PDE) inhibitors have been suggested as a possible candidate for the treatment of osteopenia, including osteoporosis. KMUP-1 is a novel xanthine derivative with inhibitory activities on the PDE 3, 4, and 5 iso-enzymes to suppress the degradation of cAMP and cGMP. This study aimed to investigate the effect of KMUP-1 on osteoblast differentiation and the underlying cellular and molecular mechanisms. Primary osteoblasts and osteoblastic MC3T3-E1 cells were examined. KMUP-1 enhanced alkaline phosphatase (ALP) activity and mineralization compared to untreated controls in primary osteoblasts and MC3T3-E1 cells. KMUP-1 also increased the mRNA expression of the osteoblastic differentiation markers, including collagen type 1a, ALP, osteocalcin, osteoprotegerin, BMP-2, and Runx2, a key transcription regulator for osteoblastic differentiation. The osteogenic effect of KMUP-1 was abolished by BMP signaling inhibitor, noggin. Furthermore, we found that KMUP-1 upregulated Smad1/5/8 phosphorylations with subsequent BRE-Luc activation confirmed by transient transfection assay. In addition, KMUP-1 inactivated glycogen synthase kinase-3ß (GSK-3ß), with associated nuclear translocation of ß-catenin. Co-treatment with H89 and KT5823, cAMP and cGMP pathway inhibitors, respectively, reversed the KMUP-1-induced activations of Smad1/5/8, ß-catenin, and Runx2. The findings demonstrate for the first time that KMUP-1 can promote osteoblast maturation and differentiation in vitro via BMP-2/Smad1/5/8 and Wnt/ß-catenin pathways. These effects are mediated, in part, by the cAMP and cGMP signaling. Thus, KMUP-1 may be a novel osteoblast activator and a potential new therapy for osteoporosis.
Assuntos
Proteína Morfogenética Óssea 2/biossíntese , Diferenciação Celular/efeitos dos fármacos , Piperidinas/administração & dosagem , Proteína Smad1/biossíntese , Proteína Smad5/biossíntese , Proteína Smad8/biossíntese , Xantinas/administração & dosagem , Animais , Proteína Morfogenética Óssea 2/genética , Calcificação Fisiológica , Linhagem Celular , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , RNA Mensageiro/biossíntese , Proteína Smad1/genética , Proteína Smad5/genética , Proteína Smad8/genética , Via de Sinalização WntRESUMO
Downregulation of miR-26b has been found in various cancers, but it has never been investigated in osteosarcoma. In this study, we demonstrated downregulation of miR-26b in osteosarcoma tissues, negatively correlated with the expression of connective tissue growth factor (CTGF) and Smad1. Luciferase reporter assay confirmed the interaction of miR-26b with the 3' untranslated regions (UTRs) of CTGF and Smad1. Transfection of miR-26b in osteosarcoma cells suppressed the expression of CTGF and Smad1, suggesting CTGF and Smad1 as direct targets of miR-26b. Overexpression of miR-26b inhibited the migration of osteosarcoma cells, which was reversed by overexpression of CTGF or Smad1. Knockdown of CTGF by small interfering RNA (siRNA) interference blocked the activation of Smad1, ERK1/2, and MMP2, which was opposite to the overexpression of CTGF. Differently, Smad1 did not significantly affect CTGF level, but mediated ERK1/2 phosphorylation and MMP2 activation. Furthermore, miR-26b inhibited lung metastasis of osteosarcoma in vivo. Our data indicated that downregulation of miR-26b in osteosarcoma elevated the levels of CTGF and Smad1, facilitating osteosarcoma metastasis.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Osteossarcoma/genética , Proteína Smad1/genética , Animais , Linhagem Celular Tumoral , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Metaloproteinase 2 da Matriz/genética , Camundongos , MicroRNAs/biossíntese , MicroRNAs/metabolismo , Metástase Neoplásica , Osteossarcoma/patologia , RNA Interferente Pequeno , Proteína Smad1/biossíntese , Proteína Smad1/metabolismoRESUMO
CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy.
Assuntos
Regeneração Óssea , Proteína Sobre-Expressa em Nefroblastoma/biossíntese , Osteoblastos/metabolismo , Regulação para Cima , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Animais , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Camundongos , Camundongos Knockout , Proteína Sobre-Expressa em Nefroblastoma/genética , Osteoblastos/patologia , Fosforilação/genética , Regiões Promotoras Genéticas/genética , Proteína Smad1/biossíntese , Proteína Smad1/genética , Proteína Smad5/biossíntese , Proteína Smad5/genética , Fator de Transcrição Sp7 , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Microtomografia por Raio-XRESUMO
The genome is under constant attack by self-produced reactive oxygen species and genotoxic reagents in the environment. Cells have evolved a DNA damage response (DDR) system to sense DNA damage, to halt cell cycle progression and repair the lesions, or to induce apoptosis if encountering irreparable damage. The best studied DDR pathways are the PIKK-p53 and PIKK-Chk1/2. Mutations in these genes encoding DDR molecules usually lead to genome instability and tumorigenesis. It is worth noting that there exist unconventional pathways that facilitate the canonical pathways or take over in the absence of the canonical pathways in DDR. This review will summarize on several unconventional pathways that participate in DDR with an emphasis on the BMP-Smad1 pathway, a known regulator of mouse development and bone remodeling.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Dano ao DNA/genética , Neoplasias/genética , Proteína Smad1/genética , Animais , Apoptose/genética , Proteínas Morfogenéticas Ósseas/genética , Remodelação Óssea/genética , Reparo do DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Proteína Smad1/biossíntese , Proteína Smad1/metabolismoRESUMO
miRNAs are endogenously expressed 18- to 25-nucleotide RNAs that regulate gene expression through translational repression by binding to a target mRNA. Recently, it has been indicated that miRNAs are closely related to osteogenesis. Our previous data suggested that miR-30 family members might be important regulators during the biomineralization process. However, whether and how they modulate osteogenic differentiation have not been explored. In this study, we demonstrated that miR-30 family members negatively regulate BMP-2-induced osteoblast differentiation by targeting Smad1 and Runx2. Evidentially, overexpression of miR-30 family members led to a decrease of alkaline phosphatase activity, whereas knockdown of them increased the activity. Then bioinformatic analysis identified potential target sites of the miR-30 family located in the 3' untranslated regions of Smad1 and Runx2. Western blot analysis and quantitative RT-PCR assays demonstrated that miR-30 family members inhibit Smad1 gene expression on the basis of repressing its translation. Furthermore, dual-luciferase reporter assays confirmed that Smad1 is a direct target of miR-30 family members. Rescue experiments that overexpress Smad1 and Runx2 significantly eliminated the inhibitory effect of miR-30 on osteogenic differentiation and provided strong evidence that miR-30 mediates the inhibition of osteogenesis by targeting Smad1 and Runx2. Also, the inhibitory effects of the miR-30 family were validated in mouse bone marrow mesenchymal stem cells. Therefore, our study uncovered that miR-30 family members are key negative regulators of BMP-2-mediated osteogenic differentiation.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , MicroRNAs/biossíntese , Família Multigênica/fisiologia , Osteoblastos/metabolismo , Biossíntese de Proteínas/fisiologia , Animais , Proteína Morfogenética Óssea 2/biossíntese , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Camundongos , MicroRNAs/genética , Osteoblastos/citologia , Proteína Smad1/biossínteseRESUMO
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by arteriovenous malformations and hemorrhages. This vascular disease results mainly from mutations in 2 genes involved in the TGF-ß pathway (ENG and ALK1) that are exclusively expressed by endothelial cells. The present study identified miR-27a and miR-205 as two circulating miRNAs differentially expressed in HHT patients. The plasma levels of miR-27a are elevated while those of miR-205 are reduced in both HHT1 and HHT2 patients compared to healthy controls. The role of miR-205 in endothelial cells was further investigated. Our data indicates that miR-205 expression displaces the TGF-ß balance towards the anti-angiogenic side by targeting Smad1 and Smad4. In line, overexpression of miR-205 in endothelial cells reduces proliferation, migration and tube formation while its inhibition shows opposite effects. This study not only suggests that detection of circulating miRNA (miR-27a and miR-205) could help for the screening of HHT patients but also provides a functional link between the deregulated expression of miR-205 and the HHT phenotype.
Assuntos
Células Endoteliais/metabolismo , MicroRNAs/fisiologia , Neovascularização Patológica/genética , Transdução de Sinais/fisiologia , Telangiectasia Hemorrágica Hereditária/genética , Transcriptoma , Fator de Crescimento Transformador beta/fisiologia , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/biossíntese , MicroRNAs/sangue , MicroRNAs/genética , Neovascularização Patológica/sangue , Neovascularização Patológica/fisiopatologia , Oligonucleotídeos Antissenso/farmacologia , Fenótipo , Curva ROC , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Transdução de Sinais/genética , Proteína Smad1/biossíntese , Proteína Smad1/genética , Proteína Smad4/biossíntese , Proteína Smad4/genética , Telangiectasia Hemorrágica Hereditária/sangue , Telangiectasia Hemorrágica Hereditária/diagnóstico , Telangiectasia Hemorrágica Hereditária/fisiopatologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
We assessed by immunohistochemistry the expression of the phosphorylated (activated) form of Smad1 and 5 (P-SMAD1/5), of Noggin and of two smooth muscle cell markers (α-SMA and SM22) in a series of human myometrium samples and in a smooth muscle cell line derived from human myometrium (HUt-SMC, PromoCell, USA). Myometrium samples were removed from two cadavers (a fetus at 26 weeks of gestation and a neonate) and from ten non-menopausal women who underwent hysterectomy for adenomyosis and leiomyoma. P-SMAD1/5 expression was never detected in myometrium (both normal and pathological specimens), but only as a nuclear positive staining in glandular and luminal epithelial cells in sections in which also the endometrial mucosa was present. Noggin was strongly expressed especially in myometrium and adenomyosis samples from non-menopausal patients in comparison to the neonatal and fetal myometrium specimens in which muscle cells were less positive. In more than 95% of HUt-SMCs, α-SMA and Desmin were co-expressed, indicating a pure smooth muscle phenotype. When progesterone was added to the culture medium, no P-SMAD1/5 expression was detected, whereas the expression Noggin and SM22, a marker of differentiated smooth muscle cells, increased by 3 fold (p=0.002) and 4.3 fold (p=0.001), respectively (p=0.002). Our results suggest that, in non-menopausal normal human myometrium, the BMP pathway might be inhibited and that this inhibition might be enhanced by progesterone, which increases the differentiation of smooth muscle cells (SM22 levels). These findings could help in the identification of new mechanisms that regulate uterine motility.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Miométrio/metabolismo , Transdução de Sinais/fisiologia , Actinas/biossíntese , Proteínas de Transporte/biossíntese , Células Cultivadas , Desmina/biossíntese , Endometriose/metabolismo , Feminino , Humanos , Histerectomia , Imuno-Histoquímica , Leiomioma/metabolismo , Proteínas dos Microfilamentos/biossíntese , Proteínas Musculares/biossíntese , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Miométrio/efeitos dos fármacos , Progesterona/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/biossíntese , Proteína Smad5/biossíntese , Neoplasias Uterinas/metabolismoRESUMO
BACKGROUND: Glomerular hypertension aggravates glomerular sclerosis by inducing growth factors, e.g., transforming growth factor-ß (TGF-ß) to mesangial matrix expansion. Smads are intracellular proteins that transmit signals from TGF-ß to nucleus, and Smads are also negatively regulated by inhibitory Smads (I-Smads), Smad6 and Smad7. However, little is known about the role of I-Smads in glomerular hypertension. We studied I-Smad expression in cultured mesangial cells subjected to mechanical stretch as an in vitro model of glomerular hypertension. METHODS: Rat mesangial cells were cultured under cyclic mechanical stretch conditions using the Flexercell Strain Unit. Phosphorylated Smad1 and Smad2 were determined by Western blots. The expression of Smad6 and Smad7 mRNAs was determined by Northern blots. Stretch-mediated I-Smad mRNAs of cells pre-treated with MAPK-ERK kinase inhibitor, U0126, were also determined. Localization of phospho-Smad1, Smad6 and Smad7 proteins in the glomerulus of Dahl salt-sensitive rats was determined by immunohistochemistry. RESULTS: Stretch stress increased phospho-Smad1 levels, and significantly decreased Smad6 mRNA to 32 % of control, and increased Smad7 mRNA to 136 % of control. U0126 significantly attenuated stretch-mediated decreases in Smad6 mRNA, but had no effect on stretch-mediated increases in Smad7 mRNA. Phospho-Smad1, Smad6 and Smad7 proteins were localized in podocytes and mesangial cells of Dahl rats. CONCLUSION: Mechanical stretch increases phospho-Smad1 levels and down-regulates Smad6 mRNA expression in mesangial cells. Stretch-mediated down-regulation of Smad6 is partially involved in ERK1/2 activation. These results indicate that glomerular hypertension might augment Smad1 signaling with concomitant attenuation of Smad6-mediated negative feedback.
Assuntos
Proteína Smad6/biossíntese , Proteína Smad7/biossíntese , Estresse Mecânico , Animais , Regulação para Baixo , Mesângio Glomerular/citologia , Mesângio Glomerular/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Dahl , Proteína Smad1/biossínteseRESUMO
PURPOSE: The exact pathogenesis of pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia (CDH) still remains unclear. Smad1, one of the bone morphogenesis protein (BMP) receptor downstream signaling proteins, plays a key role in organogenesis including lung development and maturation. Smad1 knockout mice display reduced sacculation, an important feature of pulmonary hypoplasia. Wnt inhibitor factor 1 (Wif1) is a target gene of Smad1 in the developing lung epithelial cells (LECs). Smad1 directly regulates Wif1 gene expression and blockade of Smad1 function in fetal LECs is reported to downregulate Wif1 gene expression. We designed this study to test the hypothesis that pulmonary Smad1 and Wif1 gene expression is downregulated during saccular stage of lung development in the nitrofen CDH model. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetuses were harvested on D18, and D21. Fetal lungs were dissected and divided into 2 groups: control and nitrofen (n = 9 at each time point, respectively). Pulmonary gene expression of Smad1 and Wif1 were analyzed by real-time RT-PCR. Immunohistochemistry was performed to evaluate protein expression/distribution of Smad1 and Wif1. RESULTS: The relative mRNA expression levels of Smad1 and Wif1 were significantly downregulated in the nitrofen group compared to controls on D18 and D21 (*p < 0.01, **p < 0.05). Immunoreactivity of Smad1 and Wif1 was also markedly decreased in nitrofen lungs compared to controls on D18 and D21. CONCLUSION: We provide evidence, for the first time, that the pulmonary gene expression of Smad1 and Wif1 is downregulated on D18 and D21 (saccular stage of lung development) in the nitrofen-induced hypoplastic lung. These findings suggest that the downregulation of Smad1/Wif1 gene expression may contribute to pulmonary hypoplasia in the nitrofen CDH model by retardation of lung development during saccular stage.
Assuntos
Regulação para Baixo , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Pulmão/embriologia , Prenhez , RNA Mensageiro/genética , Proteína Smad1/genética , Animais , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/biossíntese , Feminino , Hérnia Diafragmática/induzido quimicamente , Hérnia Diafragmática/complicações , Hérnia Diafragmática/genética , Hérnias Diafragmáticas Congênitas , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Pulmão/anormalidades , Pulmão/metabolismo , Pneumopatias/congênito , Pneumopatias/embriologia , Pneumopatias/genética , Organogênese/genética , Gravidez , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad1/biossínteseRESUMO
Transgenic rats with high expression of HLA-B27 and human ß(2) -microglobulin (B27TR) develop a multisystem inflammatory disease resembling human inflammatory bowel disease (IBD) and spondyloarthropaties (SpA). Tumour necrosis factor α (TNF-α) has a crucial role in sustaining chronic inflammation in the gut and joints. The aim of this work was to evaluate whether TNF-α blockade could prevent or reduce the inflammation of peripheral joints in B27TR. A first group of 9-week-old B27TR received an anti-TNF-α monoclonal antibody (mAb) or an isotypic IgG2a,k up to the age of 18 weeks. An untreated group was monitored up to the age of 18 weeks and then randomly assigned to a 9-week treatment with anti-TNF-α mAb or IgG2a,k. Each rat was monitored for clinical IBD and peripheral joint manifestations. After sacrifice the colon and hind paws were examined for macroscopical and microscopical pathological changes. Early TNF-α blockade prevented, and late treatment improved IBD signs in B27TR. Erythema, oedema, inflammatory infiltrate close to the tendons and enthesis, proliferating chondrocyte-like cells, signs of new endochondral bone ossification and bone erosion were observed in peripheral joints of four out of six IgG2a,k-treated B27TR, both at 18 and 27 weeks. Immunopositivity for phosphorylated Smad1/5/8 indicated that the process of joint remodelling was activated in B27TR. Some entheses showed chondroid nodules. Anti-TNF-α treatment reduced inflammation and preserved the enthesis organization in most animals. Occasional and transient erythema and oedema were still present in three of six of the late anti-TNF-α-treated animals. Smad1/5/8 signalling was not inhibited by late anti-TNF-α treatment. In B27TR, articular involvement follows IBD onset and develops at entheses. Early TNF-α blockade prevents the onset of IBD and consequently the development of enthesitis in peripheral joints in the B27TR model of human SpA.
Assuntos
Anticorpos Monoclonais/farmacologia , Artrite/prevenção & controle , Antígeno HLA-B27 , Doenças Inflamatórias Intestinais/terapia , Fator de Necrose Tumoral alfa/imunologia , Animais , Artrite/imunologia , Antígeno HLA-B27/genética , Antígeno HLA-B27/imunologia , Doenças Inflamatórias Intestinais/imunologia , Ratos , Ratos Transgênicos , Proteína Smad1/biossíntese , Proteína Smad1/metabolismo , Proteína Smad5/biossíntese , Proteína Smad5/metabolismo , Proteína Smad8/biossíntese , Proteína Smad8/metabolismo , Espondilite Anquilosante/prevenção & controle , Espondilite Anquilosante/terapia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Microglobulina beta-2/biossíntese , Microglobulina beta-2/genéticaRESUMO
Ossification of posterior longitudinal ligament (OPLL) is a common disease in Asian countries. Osteoblast differentiation in posterior longitudinal ligamentous fibroblast is a pathologic basis of OPLL. Nowadays, an effective pharmacotherapy for OPLL is still hunted for. YQHYRJ Recipe (YQHYRJ) is designed based on traditional Chinese medicine (TCM) theories, and previous clinic trials reported its effect on relieving syndromes of cervical spondylopathy. To clarify the YQHYRJ effect of OPLL on a cellular level, we induced mice fibroblasts from posterior longitudinal ligaments to differentiate into osteoblasts by human recombinant BMP-2, and treated them with YQHYRJ and its three sub-compounds: YQ, HY and RJ. YQHYRJ and the sub-compounds reduced the increase of fibroblast proliferation, mineralization, type I collagen secretion induced by BMP-2 via MTT, alizarin red staining and immunochemical examination. Moreover, these agents inhibited BMP-2 induced upregulation of ossification-related genes ALP, Col I and OC as well as BMP signal molecules Smad1, Smad 5 and Runx2 mRNA expression. These results suggested YQHYRJ to be effective in inhibiting osteoblast differentiation induced by BMP-2 in fibroblasts from posterior longitudinal ligament. YQHYRJ might be a promising medicine for preventing OPLL disease.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Fibroblastos/efeitos dos fármacos , Ligamentos Longitudinais/metabolismo , Osteoblastos/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Separação Celular , Cromatografia Líquida de Alta Pressão , Colágeno Tipo I/metabolismo , Corantes , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Ligamentos Longitudinais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Ossificação do Ligamento Longitudinal Posterior/metabolismo , Ossificação do Ligamento Longitudinal Posterior/patologia , Osteocalcina/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad1/biossíntese , Proteína Smad5/biossíntese , Sais de Tetrazólio , TiazóisRESUMO
Chronic kidney diseases are characterized by progressive tubulointerstitial fibrosis, and TGFbeta1 plays a crucial role in its development. Bone morphogenic protein 7 (BMP 7), another member of the TGF superfamily, antagonized the profibrotic effects of TGFbeta1, including epithelial mesenchymal transition and E-cadherin loss, in the previous studies from animal models. We investigated the effect of BMP 7 on TGFbeta1-mediated E-cadherin loss in two different transformed human adult proximal tubule epithelia. We found that BMP 7 not only failed to prevent TGFbeta1-mediated E-cadherin loss but itself downregulated E-cadherin levels and that it had an additive effect with TGFbeta1 in inducing E-cadherin loss. The downregulation of E-cadherin by BMP 7 was mediated through the Smad1/5 pathway. BMP 7-mediated E-cadherin loss was not followed by de novo alpha-smooth muscle actin (alpha-SMA) expression (a marker of myofibroblastic phenotype), which was due to the concurrent induction of Inhibitor of DNA binding 1 (Id1, a basic helix loop helix class transcriptional regulator) through a non-Smad pathway. Concurrent treatment of BMP 7 and TGFbeta1 prevented TGFbeta1-mediated alpha-SMA induction. In summary, our results suggest that E-cadherin loss, the key feature of epithelial mesenchymal transition, will not necessarily be followed by total phenotype change; rather, cells may undergo some loss of phenotypic marker in a ligand-dependent manner and participate in reparative processes. The inhibition of de novo expression of alpha-SMA could explain the antifibrotic effect of BMP 7. Id1 might play a crucial role in maintaining proximal tubule epithelial cell phenotype and its signaling regulation could be a potential therapeutic target.
Assuntos
Actinas/biossíntese , Proteína Morfogenética Óssea 7/farmacologia , Caderinas/biossíntese , Túbulos Renais Proximais/metabolismo , Rim/patologia , Músculo Liso/metabolismo , Adulto , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Fibrose/patologia , Imunofluorescência , Humanos , Proteína 1 Inibidora de Diferenciação/biossíntese , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína Smad1/biossíntese , Proteína Smad1/genética , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/fisiologia , Tubulina (Proteína)/biossínteseRESUMO
Retinoic acid can cause malformations of the developing nervous system. Smad signaling is involved in embryonic development. The current study investigated all-trans-retinoic acid (ATRA)-induced alteration of Smad expression in the developing neural tubes of mice. Pregnant mice were treated with a single dose of 50 mg/kg ATRA by oral gavage on embryonic day E7. Western immunoblotting was used to examine Smads proteins, particularly phosphorylated (p-) Smad1, total Smad1 and Smad6 in the neural tissue of the embryos on E8-E11 following treatment. Results showed that ATRA treatment significantly increased expression of both p-Smad1 and total Smad1, while Smad6 was decreased in neural tissues of ATRA-exposed embryos in utero from E8 to E11, a critical period for neural tube formation. Data suggest that disruption of Smad signaling may be involved in ATRA-induced neural tube defects.
Assuntos
Neurônios/metabolismo , Proteínas Smad/biossíntese , Tretinoína/toxicidade , Actinas/biossíntese , Animais , Western Blotting , Feminino , Expressão Gênica/efeitos dos fármacos , Camundongos , Tubo Neural/efeitos dos fármacos , Tubo Neural/crescimento & desenvolvimento , Fosforilação , Gravidez , Proteínas Smad/genética , Proteína Smad1/biossíntese , Proteína Smad1/genética , Proteína Smad6/biossíntese , Proteína Smad6/genéticaRESUMO
Mullerian-inhibiting substance (MIS), a transforming growth factor-beta family member, activates the nuclear factor-kappaB (NF-kappaB) pathway and induces the expression of B-cell translocation gene 2 (BTG2), IFN regulatory factor-1 (IRF-1), and the chemokine Gro-beta. Inhibiting NF-kappaB activation with a phosphorylation-deficient IkappaBalpha mutant abrogated MIS-mediated induction of all three genes. Expression of dominant-negative Smad1, in which serines at the COOH-terminal SSVS motif are converted to alanines, suppressed MIS-induced Smad1 phosphorylation and impaired MIS-stimulated Gro-beta promoter-driven reporter expression and Gro-beta mRNA. Suppressing Smad1 expression using small interfering RNA also mitigated MIS-induced Gro-beta mRNA, suggesting that regulation of Gro-beta expression by MIS was dependent on activation of NF-kappaB as well as Smad1. However, induction of IRF-1 and BTG2 mRNAs by MIS was independent of Smad1 activation. Characterization of kappaB-binding sequences within Gro-beta, BTG2, and IRF-1 promoters showed that MIS stimulated binding of p50 and p65 subunits to all three sites, whereas phosphorylated Smad1 (phospho-Smad1) protein was detectable only in the NF-kappaB complex bound to the kappaB site of the Gro-beta promoter. Consistent with these observations, chromatin immunoprecipitation assays showed recruitment of both phospho-Smad1 and p65 to the Gro-beta promoter in vivo, whereas p65, but not phospho-Smad1, was recruited to the BTG2 promoter. These results show a novel interaction between MIS-stimulated Smad1 and NF-kappaB signaling in which enhancement of NF-kappaB DNA binding and gene expression by phospho-Smad1 is dependent on the sequence of the kappaB consensus site within the promoter.
Assuntos
Neoplasias da Mama/metabolismo , Quimiocinas CXC/biossíntese , Glicoproteínas/farmacologia , NF-kappa B/metabolismo , Proteína Smad1/metabolismo , Hormônios Testiculares/farmacologia , Hormônio Antimülleriano , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Quimiocina CXCL2 , Quimiocinas CXC/genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Fator Regulador 1 de Interferon/biossíntese , Fator Regulador 1 de Interferon/genética , NF-kappa B/antagonistas & inibidores , Fosforilação , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad1/antagonistas & inibidores , Proteína Smad1/biossíntese , Proteína Smad1/genética , Fator de Transcrição RelA/metabolismo , Proteínas Supressoras de TumorRESUMO
BACKGROUND: Bone morphogenetic proteins (BMPs) regulate essential processes during organogenesis, and a functional understanding of these secreted proteins depends on identification of their target cells. In this study, we generate a transgenic reporter for organogenesis studies that we use to define BMP pathway activation in the developing kidney. RESULTS: Mouse strains reporting on BMP pathway activation were generated by transgenically expressing beta-galactosidase under the control of BMP responsive elements from Id1. Reporter expression corresponds well with immunoassays for pathway activation in all organs studied, validating the model. Using these reporters we have generated a detailed map of cellular targets of BMP signaling in the developing kidney. We find that SMAD dependent BMP signaling is active in collecting duct trunks, but not tips. Furthermore, glomerular endothelial cells, and proximal nephron tubules from the renal vesicle stage onward show pathway activation. Surprisingly, little activation is detected in the nephrogenic zone of the kidney, and in organ culture BMP treatment fails to activate SMAD dependent BMP signaling in nephron progenitor cells. In contrast, signaling is efficiently induced in collecting duct tips. CONCLUSION: Transgenic reporters driven by control elements from BMP responsive genes such as Id1 offer significant advantages in sensitivity and consistency over immunostaining for studies of BMP pathway activation. They also provide opportunities for analysis of BMP signaling in organ and primary cell cultures subjected to experimental manipulation. Using such a reporter, we made the surprising finding that SMAD dependent BMP signaling is inactive in nephron progenitors, and that these cells are refractory to activation by applied growth factors. Furthermore, we find that the BMP pathway is not normally active in collecting duct tips, but that it can be ectopically activated by BMP treatment, offering a possible explanation for the inhibitory effects of BMP treatment on collecting duct growth and branching.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Marcação de Genes , Genes Reporter/fisiologia , Rim/embriologia , Organogênese/fisiologia , Transdução de Sinais/fisiologia , Animais , Feminino , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Proteína Smad1/biossíntese , Proteína Smad1/genética , Proteína Smad5/biossíntese , Proteína Smad5/genética , Proteína Smad8/biossíntese , Proteína Smad8/genética , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
Although there are improvements in treatment of anorectal malformations (ARMs), patients can still develop fecal incontinence, constipation, and soiling with loss in quality of life. Recent evidence suggests that malformations in the lumbosacral spinal cord are one of the factors that affect postoperative anorectal function. However, the underlying mechanism that produces these malformations has yet to be elucidated. The bone morphogenetic proteins (BMPs) comprise a large group of highly conserved molecules that are involved in multiple processes and play important roles in the formation, development, and differentiation of the spinal cord. This study was designed to investigate the levels of BMP4 expression in the lumbosacral spinal cord in ARMs rat embryos induced by ethylenethiourea (ETU). Specifically, we assessed the association of BMP4 levels with the maldevelopment of the lumbosacral spinal cord and whether BMP4 acted through the canonical intracellular pathway in embryonic rats with ARMs. BMP4 expression was confirmed with immunohistochemical staining, RT-qPCR and western blot analyses of embryonic day (E) 16, E17, E19 and E21 embryos, moreover Smad1/5 and pSmad1/5 expression were confirmed with western blot analyses at peak time point of BMP4 expression. Our results reveal that BMP4 expression in the lumbosacral spinal cord of ARMs rat embryos is decreased at both the mRNA and protein levels and could decrease the phosphorylation of smad1/5, when compared with their expression levels in normal tissue. These results also suggest that reductions in BMP4 expression were possibly responsible for dysfunction of the lumbosacral spinal cord during late developmental stages in ARMs fetal rats. Taken together, we conclude a role for BMP4 in the pathogenesis of lumbosacral spinal cord maldevelopment in developing ARMs rats.
Assuntos
Malformações Anorretais/embriologia , Malformações Anorretais/metabolismo , Proteína Morfogenética Óssea 4/biossíntese , Região Lombossacral/embriologia , Medula Espinal/embriologia , Medula Espinal/metabolismo , Animais , Proteína Morfogenética Óssea 4/genética , Desenvolvimento Embrionário , Feminino , Fosforilação , Gravidez , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Proteína Smad1/biossíntese , Proteína Smad5/biossínteseRESUMO
AIM: Aquaporins (AQPs) are water-channels that play important roles in brain water homeostasis and cerebral edema induced by brain injury. This study aimed to investigate the relationship between AQP4, bone morphogenetic protein 4 (BMP4)/Smad1/5/8 signaling pathway and isoflurane post-conditiong, which has effects on brain edema in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: Cerebral I/R injury was induced in rats by using the middle cerebral artery occlusion (MCAO) model for 90min, followed by 24h of reperfusion. Isoflurane post-conditioning (ISO) group received 90min ischemia and underwent 1.5% isoflurane post-conditioning for 60min after initiating reperfusion. Neurobehavior, brain water content, thionine staining and 2, 3, 5-triphenyl tetrazolium chloride staining were evaluated to measure levels of brain edema and damage. Expressions of AQP4, BMP4, Smad1/5/8 and phosphorylated Smad1/5/8 were detected by using Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence (IF) staining. RESULTS: Compared with the Sham group, neurological behavior score, brain infarct volume and water content of MCAO model rats increased with reperfusion injury. However, in the ISO group, cell edema and damage of brain was significantly ameliorated (P<0.01). qRT-PCR showed less AQP4 mRNA expression in the hippocampal tissue of the ISO group than in the I/R group (P<0.01). Western blot and immunofluorescence results showed similar changes in protein levels of both groups. Related protein expressions showed expressions of BMP4 and Smad1/5/8 increased in the ISO group (P<0.01), whereas total Smad1/5/8 expression didn't change in all groups. When BMP4 inhibitor (LDN193189) was injected, expression levels of AQP4 increased and neuronal density decreased (P<0.05). By contrast, expression levels of BMP4 did not change significantly after pre-injection of AQP4 inhibitor (TGN020) (P>0.05), but neuronal density increased (P<0.05). CONCLUSION: Isoflurane post-conditioning may inhibit occurrence of brain edema and reduce cerebral I/R injury through down-regulating expression of AQP4, This process may be related to the activation of BMP4/Smad1/5/8 signaling pathway.
Assuntos
Aquaporina 4/biossíntese , Proteína Morfogenética Óssea 4/biossíntese , Isquemia Encefálica/metabolismo , Pós-Condicionamento Isquêmico/métodos , Isoflurano/administração & dosagem , Traumatismo por Reperfusão/metabolismo , Proteínas Smad Reguladas por Receptor/biossíntese , Animais , Aquaporina 4/antagonistas & inibidores , Aquaporina 4/genética , Proteína Morfogenética Óssea 4/genética , Isquemia Encefálica/terapia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Expressão Gênica , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/terapia , Masculino , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/terapia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Smad Reguladas por Receptor/genética , Proteína Smad1/biossíntese , Proteína Smad1/genética , Proteína Smad5/biossíntese , Proteína Smad5/genética , Proteína Smad8/biossíntese , Proteína Smad8/genéticaRESUMO
OBJECTIVES: Cyclooxygenase-2 (COX-2) expression is increased in colorectal cancers and has been reported to be upregulated in Peutz-Jeghers polyps. To determine whether germline and somatic loss of BMPR1A in polyps from a patient with juvenile polyposis syndrome have altered COX-2 expression, we characterized a patient with juvenile polyposis syndrome for BMPR1A germline mutations and examined the polyps for BMPR1A expression and COX-2 expression. PATIENTS AND METHODS: DNA analysis for BMPR1A was performed on a patient with juvenile polyposis syndrome. Multiple polypectomies were performed, and several polyps showed adenomatous change. Genomic DNA was extracted from polyp material for loss of heterozygosity (LOH) analyses with microsatellite markers. Immunohistochemistry was performed on sections using antibodies for BMPR1A and COX-2. RESULTS: The kindred possessed a germline BMPR1A missense mutation. In polyp domains containing cystic and adenomatous epithelium, no LOH was observed using markers near the BMPR1A locus. Immunostaining indicated decreased expression of phospho-SMAD1 (pSMAD1), functionally downstream of the mutant BMPR1A receptor in the cystic epithelium, with further reduction in adenomatous portions within the polyp. COX-2 protein, normally not expressed in the colon, was present and increased in polyp epithelium. CONCLUSIONS: Decreased expression of pSMAD1 in the cystic epithelium with further reduction in the adenomatous area, and increase in COX-2 expression within polyps from the BMPR1A heterozygote, suggest a potential mechanism for adenomatous pathogenesis in these hamartomatous polyps. This may imply that COX-2 inhibitors could be a means for chemoprevention in this syndrome.