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1.
Am J Hum Genet ; 108(2): 337-345, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33434492

RESUMO

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.


Assuntos
Transtornos 46, XX do Desenvolvimento Sexual/genética , Anormalidades Congênitas/genética , Ductos Paramesonéfricos/anormalidades , Ductos Paramesonéfricos/crescimento & desenvolvimento , Mutação , Ductos Mesonéfricos/crescimento & desenvolvimento , Adulto , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 7/genética , Códon sem Sentido , Feminino , Estudos de Associação Genética , Pleiotropia Genética , Proteínas Homeobox A10/genética , Proteínas de Homeodomínio/genética , Humanos , Fator de Transcrição PAX8/genética , Herança Paterna , Penetrância , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Proteínas Wnt/genética , Ductos Mesonéfricos/anormalidades
2.
Nat Commun ; 11(1): 4840, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973129

RESUMO

Immunotherapies revolutionized cancer treatment by harnessing the immune system to target cancer cells. However, most patients are resistant to immunotherapies and the mechanisms underlying this resistant is still poorly understood. Here, we report that overexpression of BMP7, a member of the TGFB superfamily, represents a mechanism for resistance to anti-PD1 therapy in preclinical models and in patients with disease progression while on immunotherapies. BMP7 secreted by tumor cells acts on macrophages and CD4+ T cells in the tumor microenvironment, inhibiting MAPK14 expression and impairing pro-inflammatory responses. Knockdown of BMP7 or its neutralization via follistatin in combination with anti-PD1 re-sensitizes resistant tumors to immunotherapies. Thus, we identify the BMP7 signaling pathway as a potential immunotherapeutic target in cancer.


Assuntos
Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imunoterapia/métodos , Neoplasias/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Linfócitos T CD4-Positivos , Linhagem Celular Tumoral , Feminino , Folistatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Receptor de Morte Celular Programada 1/efeitos dos fármacos , Células RAW 264.7 , Proteína Smad1/metabolismo , Transcriptoma , Microambiente Tumoral/efeitos dos fármacos
3.
J Toxicol Sci ; 45(8): 435-447, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32741896

RESUMO

The imbalance of testosterone to estradiol ratio has been related to the development of prostate diseases. Although rat models of prostate diseases induced by endocrine-disrupting chemicals (EDCs) and/or hormone exposure are commonly used to analyze gene expression profiles in the prostate, most studies utilize a single endpoint. In this study, microarray analysis was used for gene expression profiling in rat prostate tissue after exposure to EDCs and sex hormones over multiple time points (prepubertal through adulthood). We used dorsolateral prostate tissues from Sprague-Dawley rats (male offspring) and postnatally administered estradiol benzoate (EB) on postnatal days (PNDs) 1, 3, and 5, followed by treatment with additional hormones [estradiol (E) and testosterone (T)] on PNDs 90-200, as described by Ho et al. Microarray analysis was performed for gene expression profiling in the dorsolateral prostate, and the results were validated via qRT-PCR. The genes in cytokine-cytokine receptor interaction, cell adhesion molecules, and chemokines were upregulated in the EB+T+E group on PNDs 145 and 200. Moreover, early-stage downregulation of anti-inflammatory gene: bone morphogenetic protein 7 gene was observed. These findings suggest that exposure to EB, T, and E activates multiple pathways and simultaneously downregulates anti-inflammatory genes. Interestingly, these genes are reportedly expressed in prostate cancer tissues/cell lines. Further studies are required to elucidate the mechanism, including analyses using human prostate tissues.


Assuntos
Disruptores Endócrinos/toxicidade , Estradiol/análogos & derivados , Estradiol/toxicidade , Perfilação da Expressão Gênica/métodos , Expressão Gênica , Próstata/metabolismo , Puberdade , Testosterona/toxicidade , Transcriptoma , Fatores Etários , Animais , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Disruptores Endócrinos/efeitos adversos , Estradiol/efeitos adversos , Inflamação/genética , Masculino , Análise em Microsséries , Ratos Sprague-Dawley , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Testosterona/efeitos adversos
4.
Hum Genet ; 139(8): 1077-1090, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32266521

RESUMO

Our previous genome-wide association study (GWAS) for sagittal nonsyndromic craniosynostosis (sNCS) provided important insights into the genetics of midline CS. In this study, we performed a GWAS for a second midline NCS, metopic NCS (mNCS), using 215 non-Hispanic white case-parent triads. We identified six variants with genome-wide significance (P ≤ 5 × 10-8): rs781716 (P = 4.71 × 10-9; odds ratio [OR] = 2.44) intronic to SPRY3; rs6127972 (P = 4.41 × 10-8; OR = 2.17) intronic to BMP7; rs62590971 (P = 6.22 × 10-9; OR = 0.34), located ~ 155 kb upstream from TGIF2LX; and rs2522623, rs2573826, and rs2754857, all intronic to PCDH11X (P = 1.76 × 10-8, OR = 0.45; P = 3.31 × 10-8, OR = 0.45; P = 1.09 × 10-8, OR = 0.44, respectively). We performed a replication study of these variants using an independent non-Hispanic white sample of 194 unrelated mNCS cases and 333 unaffected controls; only the association for rs6127972 (P = 0.004, OR = 1.45; meta-analysis P = 1.27 × 10-8, OR = 1.74) was replicated. Our meta-analysis examining single nucleotide polymorphisms common to both our mNCS and sNCS studies showed the strongest association for rs6127972 (P = 1.16 × 10-6). Our imputation analysis identified a linkage disequilibrium block encompassing rs6127972, which contained an enhancer overlapping a CTCF transcription factor binding site (chr20:55,798,821-55,798,917) that was significantly hypomethylated in mesenchymal stem cells derived from fused metopic compared to open sutures from the same probands. This study provides additional insights into genetic factors in midline CS.


Assuntos
Proteína Morfogenética Óssea 7/genética , Craniossinostoses/genética , Variação Genética , Polimorfismo de Nucleotídeo Único/genética , Alelos , Metilação de DNA , Genes Reporter , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Íntrons/genética , Desequilíbrio de Ligação , Regiões Promotoras Genéticas/genética , Fatores de Risco
5.
J Steroid Biochem Mol Biol ; 199: 105587, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32004706

RESUMO

Chronic kidney disease (CKD) is associated with elevated circulating fibroblast growth factor 23 (FGF23), impaired renal biosynthesis of 1α,25-dihydroxyvitamin D (1α,25(OH)2D), low bone mass, and increased fracture risk. Our previous data with human mesenchymal stem cells (hMSCs) indicated that vitamin D metabolism in hMSCs is regulated as it is in the kidney and promotes osteoblastogenesis in an autocrine/paracrine manner. In this study, we tested the hypothesis that FGF23 inhibits vitamin D metabolism and action in hMSCs. hMSCs were isolated from discarded marrow during hip arthroplasty, including two subjects receiving hemodialysis and a series of 20 subjects (aged 49-83 years) with estimated glomerular filtration rate (eGFR) data. The direct in vitro effects of rhFGF23 on hMSCs were analyzed by RT-PCR, Western immunoblot, and biochemical assays. Ex vivo analyses showed positive correlations for both secreted and membrane-bound αKlotho gene expression in hMSCs with eGFR of the subjects from whom hMSCs were isolated. There was downregulated constitutive expression of αKlotho, but not FGFR1 in hMSCs obtained from two hemodialysis subjects. In vitro, rhFGF23 countered vitamin D-stimulated osteoblast differentiation of hMSCs by reducing the vitamin D receptor, CYP27B1/1α-hydroxylase, biosynthesis of 1α,25(OH)2D3, and signaling through BMP-7. These data demonstrate that dysregulated vitamin D metabolism in hMSCs may contribute to impaired osteoblastogenesis and altered bone and mineral metabolism in CKD subjects due to elevated FGF23. This supports the importance of intracellular vitamin D metabolism in autocrine/paracrine regulation of osteoblast differentiation in hMSCs.


Assuntos
Fatores de Crescimento de Fibroblastos/genética , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Insuficiência Renal Crônica/genética , Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Idoso , Idoso de 80 Anos ou mais , Proteína Morfogenética Óssea 7/genética , Diferenciação Celular/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Taxa de Filtração Glomerular , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Pessoa de Meia-Idade , Osteoblastos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptores de Calcitriol/genética , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Vitamina D/análogos & derivados , Vitamina D/biossíntese , Vitamina D/genética
6.
Mol Med Rep ; 21(1): 478-484, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31939623

RESUMO

Endothelial to mesenchymal transition (EndMT) has been confirmed to participate in several cardiovascular diseases. In addition, EndMT of circulating endothelial cells (CECs) contributes to the pathology of musculoskeletal injury. However, little is known about the molecular mechanism of CECs undergoing EndMT. In the present study, human CECs were isolated and identified using anti­CD146­coupled magnetic beads. CECs were exposed to transforming growth factor (TGF)­ß1 or TGF­ß1 + recombinant human bone morphogenetic protein 7 (rhBMP­7) or TGF­ß1 + rhBMP­7 + Smad5 antagonist Jun activation domain­binding protein 1. Vascular endothelial (VE)­cadherin and vimentin expression were detected by immunofluorescence staining in TGF­ß1­treated CECs. The expression levels of von Willebrand factor (vWF), E­selectin, VE­cadherin, vimentin, fibronectin, α smooth muscle actin (α­SMA) and Smad2/3 were detected by reverse transcription­quantitative PCR or western blot analysis. It was identified that rhBMP­7 attenuated TGF­ß1­induced endothelial cell injury. TGF­ß1 could induce the EndMT process in CECs, as confirmed by the co­expression of VE­cadherin and vimentin. TGF­ß1 significantly reduced the expression of VE­cadherin, and induced the expression of vimentin, fibronectin and α­SMA. rhBMP­7 reversed the effects of TGF­ß1 on the expression of these genes. Additionally, Smad5 antagonist reversed the effects of rhBMP­7 on TGF­ß1­induced EndMT, and upregulated rhBMP­7­inhibited Smad2/3 expression. In conclusion, TGF­ß1 could induce EndMT in CECs and rhBMP­7 may suppress this process by regulating Smad5.


Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Células Endoteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Proteína Smad5/antagonistas & inibidores , Fator de Crescimento Transformador beta1/farmacologia , Adulto , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/farmacologia , Antígeno CD146/metabolismo , Complexo do Signalossomo COP9/farmacologia , Caderinas/metabolismo , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Masculino , Peptídeo Hidrolases/farmacologia , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Proteína Smad5/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Vimentina/metabolismo
7.
Oncogene ; 39(5): 987-1003, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31591478

RESUMO

Despite intense research and clinical efforts, patients affected by advanced colorectal cancer (CRC) have still a poor prognosis. The discovery of colorectal (CR) cancer stem cell (CSC) as the cell compartment responsible for tumor initiation and propagation may provide new opportunities for the development of new therapeutic strategies. Given the reduced sensitivity of CR-CSCs to chemotherapy and the ability of bone morphogenetic proteins (BMP) to promote colonic stem cell differentiation, we aimed to investigate whether an enhanced variant of BMP7 (BMP7v) could sensitize to chemotherapy-resistant CRC cells and tumors. Thirty-five primary human cultures enriched in CR-CSCs, including four from chemoresistant metastatic lesions, were used for in vitro studies and to generate CR-CSC-based mouse avatars to evaluate tumor growth and progression upon treatment with BMP7v alone or in combination with standard therapy or PI3K inhibitors. BMP7v treatment promotes CR-CSC differentiation and recapitulates the cell differentiation-related gene expression profile by suppressing Wnt pathway activity and reducing mesenchymal traits and survival of CR-CSCs. Moreover, in CR-CSC-based mouse avatars, BMP7v exerts an antiangiogenic effect and sensitizes tumor cells to standard chemotherapy regardless of the mutational, MSI, and CMS profiles. Of note, tumor harboring PIK3CA mutations were affected to a lower extent by the combination of BMP7v and chemotherapy. However, the addition of a PI3K inhibitor to the BMP7v-based combination potentiates PIK3CA-mutant tumor drug response and reduces the metastatic lesion size. These data suggest that BMP7v treatment may represent a useful antiangiogenic and prodifferentiation agent, which renders CSCs sensitive to both standard and targeted therapies.


Assuntos
Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/farmacologia , Neoplasias Colorretais/patologia , Mutação , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Humanos , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Turk Neurosurg ; 30(3): 371-376, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31452178

RESUMO

AIM: To investigate the effects of different therapeutic radiation doses on the prevalence of neural tube defects (NTDs) in chick embryos and bone morphogenetic protein (BMP) 4 and BMP7 expression levels. MATERIAL AND METHODS: The chick embryos (n=143) were derived from fertile, specific pathogen-free eggs of domestic fowl. The presence of NTDs was analyzed using a stereomicroscope, and BMP4 and BMP7 expression levels were assessed by immunohistochemical staining. The chick embryos were divided into five groups: control (no radiation exposure) (n=23), exposure to thorax computerized tomography (CT) (n=30); exposure to abdominopelvic CT (n=30), exposure to cranium CT (n=30), and exposure to brain perfusion CT (n=30). RESULTS: The prevalence of NTDs and BMP4 and BMP7 expression levels in the different groups were compared. In the cranium CT dose group, both the NTD prevalence (20%, p=0.002) and BMP7 (p=0.031) expression levels were significantly higher than those in the other groups. However, none of the medical doses of irradiation altered BMP4 expression levels (p=0.242). No NTDs were detected in the thorax CT and abdominopelvic CT groups. CONCLUSION: Exposure to irradiation at cranium CT doses may induce the development of NTDs and increase BMP7 expression. Dose radiation exposure using thorax CT and abdominopelvic CT protocols does not appear to induce NTDs.


Assuntos
Proteína Morfogenética Óssea 4/biossíntese , Proteína Morfogenética Óssea 7/biossíntese , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/radioterapia , Doses de Radiação , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 7/genética , Embrião de Galinha , Expressão Gênica , Tubo Neural/diagnóstico por imagem , Tubo Neural/efeitos da radiação , Defeitos do Tubo Neural/diagnóstico por imagem , Defeitos do Tubo Neural/genética
9.
Nat Commun ; 10(1): 5364, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31792207

RESUMO

DNA methylation regulates cell type-specific gene expression. Here, in a transgenic mouse model, we show that deletion of the gene encoding DNA methyltransferase Dnmt3a in hypothalamic AgRP neurons causes a sedentary phenotype characterized by reduced voluntary exercise and increased adiposity. Whole-genome bisulfite sequencing (WGBS) and transcriptional profiling in neuronal nuclei from the arcuate nucleus of the hypothalamus (ARH) reveal differentially methylated genomic regions and reduced expression of AgRP neuron-associated genes in knockout mice. We use read-level analysis of WGBS data to infer putative ARH neural cell types affected by the knockout, and to localize promoter hypomethylation and increased expression of the growth factor Bmp7 to AgRP neurons, suggesting a role for aberrant TGF-ß signaling in the development of this phenotype. Together, these data demonstrate that DNA methylation in AgRP neurons is required for their normal epigenetic development and neuron-specific gene expression profiles, and regulates voluntary exercise behavior.


Assuntos
Metilação de DNA , Neurônios/metabolismo , Condicionamento Físico Animal , Adiposidade , Animais , Comportamento Animal , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Feminino , Hipotálamo/citologia , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais
10.
Cell Death Dis ; 10(11): 852, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31699966

RESUMO

The extracellular matrix (ECM) is known to regulate tissue development and cell morphology, movement, and differentiation. SPARCL1 is an ECM protein, but its role in mouse cell differentiation has not been widely investigated. The results of western blotting and immunofluorescence showed that SPARCL1 is associated with the repair of muscle damage in mice and that SPARCL1 binds to bone morphogenetic protein 7 (BMP7) by regulating BMP/transforming growth factor (TGF)-ß cell signaling. This pathway promotes the differentiation of C2C12 cells. Using CRISPR/Cas9 technology, we also showed that SPARCL1 activates BMP/TGF-ß to promote the differentiation of C2C12 cells. BMP7 molecules were found to interact with SPARCL1 by immunoprecipitation analysis. Western blotting and immunofluorescence were performed to verify the effect of BMP7 on C2C12 cell differentiation. Furthermore, SPARCL1 was shown to influence the expression of BMP7 and activity of the BMP/TGF-ß signaling pathway. Finally, SPARCL1 activation was accompanied by BMP7 inhibition in C2C12 cells, which confirmed that SPARCL1 affects BMP7 expression and can promote C2C12 cell differentiation through the BMP/TGF-ß pathway. The ECM is essential for muscle regeneration and damage repair. This study intends to improve the understanding of the molecular mechanisms of muscle development and provide new treatment ideas for muscle injury diseases.


Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Músculo Esquelético/citologia , Mioblastos/citologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteína Morfogenética Óssea 7/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas da Matriz Extracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/genética
11.
Pharmacology ; 104(5-6): 352-358, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31618732

RESUMO

We evaluated the effect of microRNA (miR)-9 inhibition on fracture healing in a rat model of femoral fracture. The rats were divided into sham, negative control and miR-9 inhibitor groups. The miR-9 inhibitor group received 30 pmol/mL inhibitor intrathecally for 8 consecutive weeks following surgery-induced femoral fracture. The effect of miR-9 inhibition on fracture healing was estimated by determining the bone mineral density (BMD) and by performing X-ray analysis of the fractured bone. The serum levels of markers of bone formation were estimated by enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction, and western blotting and immunohistochemical analysis were performed to assess the effect of miR-9 inhibition on fracture healing. The BMD at the fracture site was significantly higher in the miR-9 inhibitor group than in the negative control group. Inhibition of miR-9 blocked the fracture gap and resulted in new callus formation at the fracture site. The serum levels of osteocalcin and bone GLA protein were increased and that of alkaline phosphatase was decreased by inhibition of miR-9 compared to levels in the negative control. However, inhibition of miR-9 significantly increased the mRNA levels of runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 7 (BMP-7) in the bone tissue at the fracture site compared to the negative control group; this result was confirmed by western blotting. In conclusion, -miR-9 inhibition enhanced fracture healing by modulating the BMP-7/Runx2 signalling pathway in a rat model of femoral fracture.


Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Consolidação da Fratura/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Animais , Proteína Morfogenética Óssea 7/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fêmur/efeitos dos fármacos , Fêmur/metabolismo , Fêmur/patologia , Masculino , Osteogênese/efeitos dos fármacos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
12.
Life Sci ; 238: 116957, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31655195

RESUMO

Epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition in renal tubular epithelial cells are critical to diabetic nephropathy (DN) pathogenesis, but the underlying mechanisms remain undefined. Bone morphogenetic protein 7 (BMP-7) inhibits EMT and ECM accumulation in renal tubular epithelial cells cultured in presence of high glucose. Meanwhile, miRNA-21 (miR-21) downregulates Smad7, promoting EMT and ECM deposition. However, the association of BMP-7 with miR-21/Smad7 in DN is unknown. Here, NRK-52E cells incubated in presence of high glucose and STZ-induced C57BL diabetic mice were considered in vitro and in vivo models of DN, respectively. In both models, BMP-7 (mRNA/protein) amounts were decreased as well as Smad7 protein expression, while miR-21 expression and TGF-ß1/Smad3 pathway activation were enhanced, accompanied by enhanced EMT and ECM deposition. Further, addition of BMP-7 human recombinant cytokine (rhBMP-7) and injection of the BMP-7 overexpression plasmid in diabetic mice markedly downregulated miR-21 and upregulated Smad7, reduced Smad3 activation without affecting TGF-ß1 amounts, and prevented EMT and ECM accumulation. MiR-21 overexpression in the in vitro model downregulated Smad7, promoted EMT and ECM accumulation without affecting BMP-7 amounts, and miR-21 downregulation reversed it. By interfering with BMP-7 and miR-21 expression in high glucose conditions, miR-21 amounts and Smad3 phosphorylation were further decreased. Smad7 was then upregulated, and EMT and ECM deposition were inhibited; these effects were reversed after miR-21 overexpression. These findings suggest that BMP-7 decreases renal fibrosis in DN by regulating miR-21/Smad7 signaling, providing a theoretical basis for the development of novel and effective therapeutic drugs for DN.


Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/complicações , Fibrose/prevenção & controle , Regulação da Expressão Gênica , Túbulos Renais/metabolismo , MicroRNAs/antagonistas & inibidores , Animais , Proteína Morfogenética Óssea 7/genética , Células Cultivadas , Transição Epitelial-Mesenquimal , Matriz Extracelular/metabolismo , Fibrose/etiologia , Fibrose/patologia , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Fosforilação , Transdução de Sinais , Proteína Smad7/genética , Proteína Smad7/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
13.
Elife ; 82019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31566563

RESUMO

BMP7/BMP2 or BMP7/BMP4 heterodimers are more active than homodimers in vitro, but it is not known whether these heterodimers signal in vivo. To test this, we generated knock in mice carrying a mutation (Bmp7R-GFlag) that prevents proteolytic activation of the dimerized BMP7 precursor protein. This mutation eliminates the function of BMP7 homodimers and all other BMPs that normally heterodimerize with BMP7. While Bmp7 null homozygotes are live born, Bmp7R-GFlag homozygotes are embryonic lethal and have broadly reduced BMP activity. Furthermore, compound heterozygotes carrying the Bmp7R-G allele together with a null allele of Bmp2 or Bmp4 die during embryogenesis with defects in ventral body wall closure and/or the heart. Co-immunoprecipitation assays confirm that endogenous BMP4/7 heterodimers exist. Thus, BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian development, which may explain why mutations in either Bmp4 or Bmp7 lead to a similar spectrum of congenital defects in humans.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Desenvolvimento Embrionário , Multimerização Proteica , Animais , Proteína Morfogenética Óssea 7/genética , Técnicas de Introdução de Genes , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo
14.
Circ Res ; 125(9): 834-846, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31495264

RESUMO

RATIONALE: Pathogenic variations in the lamin gene (LMNA) cause familial dilated cardiomyopathy (DCM). LMNA insufficiency caused by LMNA pathogenic variants is believed to be the basic mechanism underpinning LMNA-related DCM. OBJECTIVE: To assess whether silencing of cardiac Lmna causes DCM and investigate the role of Yin Yang 1 (Yy1) in suppressing Lmna DCM. METHODS AND RESULTS: We developed a Lmna DCM mouse model induced by cardiac-specific Lmna short hairpin RNA. Silencing of cardiac Lmna induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of Yy1 suppressed Lmna DCM and cardiac fibrosis by inducing Bmp7 expression and preventing upregulation of Ctgf. Knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. However, upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of Yy1 regulated Bmp7 and Ctgf reporter activities and modulated Bmp7 and Ctgf gene expression in cardiomyocytes. Downregulation of Ctgf inhibited TGF-ß (transforming growth factor-ß)/Smad signaling in DCM hearts. Regulation of both Bmp7 and Ctgf further suppressed TGFß/Smad signaling. In addition, co-modulation of Bmp7 and Ctgf reduced CD3+ T cell numbers in DCM hearts. CONCLUSIONS: Our findings demonstrate that upregulation of Yy1 or co-modulation of Bmp7 and Ctgf offer novel therapeutic strategies for the treatment of DCM caused by LMNA insufficiency.


Assuntos
Proteína Morfogenética Óssea 7/biossíntese , Cardiomiopatias/metabolismo , Cardiomiopatias/prevenção & controle , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Fator de Transcrição YY1/biossíntese , Animais , Proteína Morfogenética Óssea 7/genética , Cardiomiopatias/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Endotélio Vascular/metabolismo , Fibrose/genética , Fibrose/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição YY1/genética
15.
Int J Biochem Cell Biol ; 116: 105613, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31539631

RESUMO

Renal tubular epithelial-mesenchymal transition (EMT) is the main pathological change in diabetic renal tubulointerstitial fibrosis. Mounting evidence indicates that the inhibitor of differentiation 2 (Id2) protein acts as a negative regulatory factor in organ fibrosis and can inhibit or reverse the process of fibrosis. However, its specific regulatory mechanism is not clear. Diabetes mellitus (DM) rat models were established by injecting rats with streptozotocin and sacrificing them after 16 weeks. Rat renal tubular epithelial cells (NRK-52E) were cultured with normal and high glucose. Immunohistochemical analysis, double immunofluorescence staining, co-immunoprecipitation, Western blot analysis, and real-time polymerase chain reaction were used to determine the expression of Id2, Twist, Smad1/5/8, E-cadherin, α-smooth muscle actin (α-SMA), and collagen Ⅳ. The results showed that bone morphogenetic protein-7 (BMP-7) upregulated the expression of Id2 against high-glucose-induced EMT and extracellular matrix secretion. Moreover, only the simultaneous knockdown of Smad1, Smad5, and Smad8 downregulated the expression of Id2, which was not altered by the individual knockdown of Smad1, Smad5, and Smad8. Basic helix-loop-helix (bHLH) transcription factors were essential for Id2 to regulate the role of downstream target genes, and Twist was a bHLH transcription factor. Therefore, the expression of Twist was examined in this study. Twist was found to be highly expressed in the kidney of DM rats and renal tubular epithelial cells cultured with high glucose. The overexpression of Id2 did not alter the expression of Twist, but the interaction between Id2 and Twist was enhanced. In conclusion, the data showed the specific mechanism underlying Id2 negative regulation in diabetic renal tubulointerstitial fibrosis.


Assuntos
Proteína Morfogenética Óssea 7/genética , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/genética , Proteína 2 Inibidora de Diferenciação/genética , Nefrite Intersticial/genética , Proteínas Smad/genética , Proteína 1 Relacionada a Twist/genética , Actinas/genética , Actinas/metabolismo , Animais , Proteína Morfogenética Óssea 7/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/induzido quimicamente , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Matriz Extracelular/química , Matriz Extracelular/efeitos dos fármacos , Regulação da Expressão Gênica , Glucose/farmacologia , Proteína 2 Inibidora de Diferenciação/metabolismo , Masculino , Nefrite Intersticial/induzido quimicamente , Nefrite Intersticial/metabolismo , Nefrite Intersticial/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas Smad/antagonistas & inibidores , Proteínas Smad/metabolismo , Estreptozocina/administração & dosagem , Proteína 1 Relacionada a Twist/metabolismo
16.
Mol Immunol ; 114: 251-259, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31398664

RESUMO

BACKGROUND: Fracture healing is a complex process, and patients with fracture will undergo non-union or compromised regeneration. MicroRNA (miR)-342-5p is a Notch downstream molecule, and its roles in fracture healing remain unclear. We aimed to explore the functional roles of miR-342-5p in osteoblasts as well as the underlying mechanisms. METHODS: The expression of miR-342-5p in differentiation of MC3T3-E1 cells or hMSCs was examined by quantitative reverse transcription PCR (qRT-PCR). The effects of aberrantly expressed miR-342-5p on cell proliferation, apoptosis, migration, and expressions of proteins associated with proliferation and osteogenic differentiation were determined by Cell Counting Kit-8, trypan blue staining, flow cytometry, Transwell assay, Western blot and qRT-PCR assays, respectively. The downstream factor and the target genes of miR-342-5p as well as the involvements of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were finally assessed. RESULTS: miR-342-5p level was decreased during differentiation of MC3T3-E1 cells or hMSCs. After cell transfection, miR-342-5p overexpression significantly reduced cell viability, induced apoptosis, inhibited proliferation, migration and differentiation, and down-regulated Bmp7 expression. Subsequent experiments showed the effects of miR-342-5p inhibition on MC3T3-E1 cells were abrogated by Bmp7 knockdown. Additionally, COL4A6 and Bmp2 were predicated as target genes of miR-342-5p. Finally, phosphorylated levels of MEK and ERK were increased by miR-342-5p inhibition via up-regulating Bmp7 expression. CONCLUSION: miR-342-5p inhibition promoted proliferation, migration and differentiation of osteoblasts via regulating Bmp7, along with activation of the MEK/ERK pathway.


Assuntos
Proteína Morfogenética Óssea 7/genética , Diferenciação Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , MicroRNAs/genética , Osteoblastos/patologia , Células 3T3 , Adulto , Animais , Apoptose/genética , Linhagem Celular , Sobrevivência Celular/genética , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/genética , Osteogênese/genética , Transdução de Sinais/genética , Regulação para Cima/genética , Adulto Jovem
17.
J Endocrinol ; 243(2): 97-110, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31394500

RESUMO

Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor-ß (TGF-ß) family, plays pivotal roles in energy expenditure. However, whether and how BMP7 regulates hepatic insulin sensitivity is still poorly understood. Here, we show that hepatic BMP7 expression is reduced in high-fat diet (HFD)-induced diabetic mice and palmitate (PA)-induced insulin-resistant HepG2 and AML12 cells. BMP7 improves insulin signaling pathway in insulin resistant hepatocytes. On the contrary, knockdown of BMP7 further impairs insulin signal transduction in PA-treated cells. Increased expression of BMP7 by adenovirus expressing BMP7 improves hyperglycemia, insulin sensitivity and insulin signal transduction. Furthermore, BMP7 inhibits mitogen-activated protein kinases (MAPKs) in both the liver of obese mice and PA-treated cells. In addition, inhibition of MAPKs recapitulates the effects of BMP7 on insulin signal transduction in cultured hepatocytes treated with PA. Activation of p38 MAPK abolishes the BMP7-mediated upregulation of insulin signal transduction both in vitro and in vivo. Together, our results show that hepatic BMP7 has a novel function in regulating insulin sensitivity through inhibition of MAPKs, thus providing new insights into treating insulin resistance-related disorders such as type 2 diabetes.


Assuntos
Proteína Morfogenética Óssea 7/genética , Regulação da Expressão Gênica , Resistência à Insulina/genética , Fígado/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Animais , Proteína Morfogenética Óssea 7/metabolismo , Linhagem Celular , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Células Hep G2 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Obesidade/genética , Obesidade/metabolismo
18.
J Assist Reprod Genet ; 36(6): 1127-1133, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31065945

RESUMO

PURPOSE: To evaluate the relationship of clinical pregnancy rates with bone morphogenetic proteins 2-4-7 (BMP 2, 4, 7), growth differentiation factor 9 (GDF 9), and Emmprin levels in follicular fluid of intracytoplasmic sperm injection patients. METHODS: Follicular fluid of 77 patients who underwent ICSI procedure was collected during the oocyte retrieval procedure. And follicular fluid levels of BMP 2, BMP 4, BMP 7, GDF 9, and Emmprin (Basigin) were measured and compared for clinical pregnancy rates. RESULTS: Follicular levels of BMP 4 was significantly higher whereas Emmprin levels were lower in patients who had achieved clinically diagnosed pregnancy compared with those who did not achieve clinical pregnancy after ICSI procedure (P = 0.007 and P = 0.035, respectively). BMP 2, BMP 7, and GDF 9 levels were comparable for both groups. CONCLUSION: Clinical pregnancy rates after ICSI may be associated with follicular fluid levels of Emmprin and BMP 4. Follicular levels of Emmprin and BMP 4 can be used as a marker (as markers for predicting ICSI outcomes) for a better ICSI outcome.


Assuntos
Basigina/genética , Proteína Morfogenética Óssea 4/genética , Infertilidade Feminina/genética , Taxa de Gravidez , Adulto , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 7/genética , Feminino , Fertilização In Vitro , Líquido Folicular/metabolismo , Fator 9 de Diferenciação de Crescimento/genética , Humanos , Infertilidade Feminina/epidemiologia , Infertilidade Feminina/patologia , Masculino , Recuperação de Oócitos , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Indução da Ovulação/métodos , Gravidez , Injeções de Esperma Intracitoplásmicas/métodos
19.
J Cell Biochem ; 120(10): 17512-17519, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31127659

RESUMO

INTRODUCTION: Diabetic nephropathy (DN) is one of the critical complications of diabetes mellitus and the main cause of chronic renal dysfunction. The pathogenic mechanism causing the disease remains unclear and there is a lack of effective treatment methods so novel strategies are needed for DN management. The aim of this study, therefore, is to evaluate the effect of liraglutide as glucagon-like peptide-1 analogue and its underlying mechanisms on induced DN in rats MATERIALS AND METHODS: Sixty rats were divided into control group, diabetic group, and liraglutide-treated group. At the end of experiment, renal CTGF and BMP-7 messeger RNA expression were determined. Blood sugar, serum urea, and creatinine were measured. Also, histopathological changes were studied. RESULTS: Liraglutide can improve renal alterations associated with diabetes as it reduced CTGF expression and increased BMP-7 expression. In the same time, it could improve histopathological changes and renal function tests. CONCLUSION: These findings influence the beneficial use of liraglutide for the management of DN in patients with diabetes mellitus.


Assuntos
Proteína Morfogenética Óssea 7/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Nefropatias Diabéticas/tratamento farmacológico , Liraglutida/farmacologia , Animais , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/genética , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Ratos
20.
Anim Reprod Sci ; 204: 183-192, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30962038

RESUMO

Although it has been investigated for many years, the physiological processes regulating prolificacy in sheep remains unclear because of regulation by many genes. To better understand the effects of three single nucleotide polymorphisms (SNPs) comprising g.48462350C>T in BMP2, g.58171856C>G and g.58171886A>C in BMP7, a population genetic analysis was conducted using data obtained from genotyping in 768 sheep from six breeds (three polytocous and three monotocous). The results indicate that all the sheep breeds were considered to conform to the Hardy-Weinberg equilibrium (P > 0.05). The associations of these three SNPs with litter size in 384 Small Tail Han sheep were analyzed, therefore, and found to be correlated with fecundity as assessed by mean litter size (P < 0.05). Bioinformatic analysis indicated there was a transmembrane domain change that occurred after a mutation in BMP2 at g.48462350C>T, and changes involving transcription factors such as USF1, USF2 and INMS1 in the BMP7 promoter region might be involved in greater sheep prolificacy.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Tamanho da Ninhada de Vivíparos/genética , Ovinos/genética , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 7/genética , Biologia Computacional , Feminino , Regulação da Expressão Gênica/fisiologia , Genótipo , Homozigoto , Polimorfismo de Nucleotídeo Único
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