scholarly journals Effects of MiR-375-BMPR2 as a Key Factor Downstream of BMP15/GDF9 on the Smad1/5/8 and Smad2/3 Signaling Pathways

2018 ◽  
Vol 46 (1) ◽  
pp. 213-225 ◽  
Author(s):  
Chang Liu ◽  
Bao Yuan ◽  
Hongyan Chen ◽  
Mingqiang Xu ◽  
Xulei Sun ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Cell Counting ◽  
Type I ◽  
Type Ii ◽  
Expression Levels ◽  
Smad Signaling ◽  
Proliferation And Apoptosis ◽  
Smad Signaling Pathway

Background/Aims: Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), which are secreted by oocytes, are important regulators of follicular growth and development and ovarian function. These two factors can regulate the proliferation and apoptosis of cumulus cells via modulation of the Smad signaling pathway. Studies have shown that BMP15 and GDF9 can affect the level of miR-375, whereas the target gene of miR-375 is BMPR2, the type II receptor of BMP15 and GDF9. However, whether or how the BMP15/ GDF9-miR-375-BMPR2 pathway affects the proliferation and apoptosis of bovine cumulus cells through regulation of the Smad signaling pathway remains unclear. Methods: In this study, cumulus cells were first obtained from cumulus-oocyte complexes (COCs). Appropriate concentrations of BMP15 and GDF9 were added during the in vitro culture process. Cell Counting Kit-8 (CCK-8) analyses and flow cytometry were used to determine the effects of BMP15/GDF9 on bovine cumulus cells proliferation and apoptosis. Subsequently, miR-375 mimics, miR-375 inhibitor and BMPR2 siRNA were synthesized and used for transfection experiments. Western Blot analysis was used to detect changes before and after transfection in the expression levels of the BMP15/GDF9 type I receptors ALK4, ALK5 and ALK6; the phosphorylation levels of Smad2/3 and Smad1/5/8, which are key signaling pathway proteins downstream of BMP15/GDF9; the expression levels of PTX3, HAS2 and PTGS2, which are key genes involved in cumulus cells proliferation; and Bcl2/Bax, which are genes involved in apoptosis. Results: The addition of 100 ng/mL BMP15 or 200 ng/mL GDF9 or the combined addition of 50 ng/mL BMP15 and 100 ng/mL GDF9 effectively inhibited bovine cumulus cell apoptosis and promoted cell proliferation. BMP15/GDF9 negatively regulated miR-375 expression and positively regulated BMPR2 expression. High levels of miR-375 and inhibition of BMPR2 resulted in increased expression of ALK4 and decreased expression of PTX3, HAS2 and PTGS2, whereas miR-375 inhibition resulted in the opposite results. BMP15 and GDF9 significantly activated the levels of p-Smad2/3 and p-Smad1/5/8, whereas miR-375 inhibited the levels of p-Smad2/3 and p-Smad1/5/8 by negatively regulating BMPR2 and also led to apoptosis. Conclusion: BMP15 and GDF9 have synergistic effects and can act through miR-375 to affect the expression levels of type I receptor ALK4 and type II receptor BMPR2 and the activation of Smad signaling pathway, which subsequently affected the proliferation, spread and apoptosis of cumulus cells.

scholarly journals Roles of gene transcription and PKA subtype activation in maturation of murine oocytes

Reproduction ◽  
2002 ◽  
pp. 799-806 ◽  
Author(s):  
KF Rodriguez ◽  
RM Petters ◽  
AE Crosier ◽  
CE Farin
Keyword(s):  
Gene Transcription ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Type I ◽  
Type Ii ◽  
Germinal Vesicle Breakdown ◽  
Series Of Experiments ◽  
And Control ◽  
Alpha Amanitin

The aims of this study were to examine the role of transcription and the coincident involvement of type I and type II protein kinase A (PKA) in the resumption of meiosis in murine cumulus-oocyte complexes (COCs) using the transcriptional inhibitors 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin. The first series of experiments was designed to: (i) characterize the role of transcription in gonadotrophin-mediated and spontaneous maturation of murine oocytes; (ii) examine the roles of specific gonadotrophins (FSH versus hCG) and cumulus cells in transcriptionally mediated oocyte maturation; and (iii) determine the reversibility of the transcriptional arrest of meiosis. In the presence of FSH, transcriptional inhibitors arrested germinal vesicle breakdown (GVBD) (DRB: 2 +/- 2% and control: 76 +/- 2%; alpha-amanitin: 4 +/- 4% and control: 70 +/- 4%). Furthermore, cumulus cells were required for transcriptional inhibitors to arrest GVBD (DRB with cumulus cells: 0 +/- 15%; DRB without cumulus cells: 94 +/- 13%; alpha-amanitin with cumulus cells: 15 +/- 2%; alpha-amanitin without cumulus cells: 99 +/- 2%). Thus, in mice, FSH-mediated GVBD uses a transcriptional mechanism, which probably occurs within the cumulus cell compartment. In a second series of experiments, the role of transcription in mediating the resumption of meiosis after activation of either type I or type II PKA was examined. Activation of type I PKA in murine COCs resulted in an arrest of GVBD that was independent of a transcriptional event (with DRB: 7 +/- 9% GVBD; without DRB: 11 +/- 9% GVBD). In contrast, activation of type II PKA resulted in a resumption of meiosis, which required the occurrence of gene transcription (with DRB: 12 +/- 9% GVBD; without DRB: 80 +/- 9% GVBD). As FSH binding to cumulus cells activates the PKA second messenger system, our results indicate that, in cultured murine COCs, FSH binding to cumulus cells results in the activation of type II PKA, which, in turn, mediates a downstream transcriptional event required for the initiation of GVBD.

scholarly journals HuangQi Decoction Ameliorates Renal Fibrosis via TGF-β/Smad Signaling Pathway In Vivo and In Vitro

2016 ◽  
Vol 38 (5) ◽  
pp. 1761-1774 ◽  
Author(s):  
Jie Zhao ◽  
Li Wang ◽  
Ai-li Cao ◽  
Ming-Qian Jiang ◽  
Xia Chen ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Ureteral Obstruction ◽  
Interstitial Fibrosis ◽  
Unilateral Ureteral Obstruction ◽  
Type I ◽  
Renal Interstitial Fibrosis ◽  
Smad Signaling ◽  
Smad Signaling Pathway ◽  
Tgf Β1

Objective: Traditional Chinese Medicine compound HuangQi decoction is widely used in clinical treatment of chronic kidney disease, but its role on renal interstitial fibrosis and the underlying mechanism remains unclear. The aim of this study is to investigate the effect of HuangQi decoction on renal interstitial fibrosis and its association with the TGF-β/Smad signaling pathway Methods: A total of 120 C57/BL mice were randomly divided into six groups: sham group, sham plus high-dose HuangQi decoction (1.08g/kg) group, unilateral ureteral obstruction (UUO) model group, and UUO model plus low to high doses of HuangQi decoction (0.12g/kg, 0.36g/kg and 1.08g/kg respectively) groups. Animals were sacrificed 14 days after the administration and ipsilateral kidney tissue was sampled for pathologic examinations. Immunohistochemistry, PCR and western blot were used to detect the expressions of related molecules in the TGF-β/Smad signaling pathway. TGF-β1 was used in in vitro experiments to induce human kidney proximal tubule epithelial cells (HK2). Results: HuangQi decoction improved ipsilateral kidney fibrosis in UUO mice and downregulated the expressions of TGF-β1, TβRI, TβRII, Smad4, Smad2/3, P-Smad2/3, α-SMA, collagen type I, III and IV in a dose-dependent manner while upregulated the expression of Smad7 in the same fashion. Similar results were found in in vitro studies. Conclusion: The protective effect of HuangQi decoction for unilateral ureteral obstruction kidney damage in mice was mediated by downregulating the TGF-β/Smad signaling pathway.

scholarly journals Pilose Antler Peptide-3.2KD Ameliorates Adriamycin-Induced Myocardial Injury Through TGF-β/SMAD Signaling Pathway

2021 ◽  
Vol 8 ◽  
Author(s):  
Yan Xu ◽  
Xiaobo Qu ◽  
Jia Zhou ◽  
Guangfu Lv ◽  
Dong Han ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Myocardial Fibrosis ◽  
Myocardial Injury ◽  
Pathological Examination ◽  
Protective Mechanism ◽  
Protective Effects ◽  
Expression Levels ◽  
Smad Signaling ◽  
Protein Levels ◽  
Smad Signaling Pathway

Adriamycin (ADR)-based combination chemotherapy is the standard treatment for some patients with tumors in clinical, however, long-term application can cause dose-dependent cardiotoxicity. Pilose Antler, as a traditional Chinese medicine, first appeared in the Han Dynasty and has been used to treat heart disease for nearly a thousand years. Previous data revealed pilose antler polypeptide (PAP, 3.2KD) was one of its main active components with multiple biological activities for cardiomyopathy. PAP-3.2KD exerts protective effects againt myocardial fibrosis. The present study demonstrated the protective mechanism of PAP-3.2KD against Adriamycin (ADR)-induced myocardial injury through using animal model with ADR-induced myocardial injury. PAP-3.2KD markedly improved the weight increase and decreased the HW/BW index, heart rate, and ST height in ADR-induced groups. Additionally, PAP-3.2KD reversed histopathological changes (such as disordered muscle bundles, myocardial fibrosis and diffuse myocardial cellular edema) and scores of the heart tissue, ameliorated the myocardial fibrosis and collagen volume fraction through pathological examination, significantly increased the protein level of Bcl-2, and decreased the expression levels of Bax and caspase-3 in myocardial tissue by ELISA, compared to those in ADR-induced group. Furthermore, ADR stimulation induced the increased protein levels of TGF-β1 and SMAD2/3/4, the increased phosphorylation levels of SMAD2/3 and the reduced protein levels of SMAD7. The expression levels of protein above in ADR-induced group were remarkably reversed in PAP-3.2KD-treated groups. PAP-3.2KD ameliorated ADR-induced myocardial injury by regulating the TGF-β/SMAD signaling pathway. Thus, these results provide a strong rationale for the protective effects of PAP against ADR-induced myocardial injury, when ADR is used to treat cancer.

scholarly journals Bioflavonoid Galangin Suppresses Hypertrophic Scar Formation by the TGF-β/Smad Signaling Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Zha Ru ◽  
Ying Hu ◽  
Shenghua Huang ◽  
Li Bai ◽  
Kun Zhang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Hypertrophic Scar ◽  
Healing Process ◽  
Scar Formation ◽  
Type Iii Collagen ◽  
Type I ◽  
Smad Signaling ◽  
Type Iii ◽  
Smad Signaling Pathway ◽  
Tgf Β1

Background. Hypertrophic scar (HS) is a benign fibroproliferative skin disease resulting from an aberrant wound healing process and can cause aesthetic and functional damage to patients. Currently, there is no ideal treatment to treat this disease. Galangin, a natural active bioflavonoid compound, is suggested to inhibit fibrosis and proliferation in certain cells. Methods. In this study, we found Galangin could attenuate abnormal scar formation in an HS rabbit ear model. Additionally, the HE staining shows Galangin reduced scar elevation index (SEI) and Masson’s trichrome staining changed collagen deposition. Results. The expressions of type I collagen, type III collagen, and TGF-β1 were much lower under a proper dose of Galangin treatment, and Smad7 expression was also enhanced, which are examined by real-time PCR, immunohistochemistry, and western blot. Conclusion. Our data indicated that Galangin can alleviate dermal scarring via the TGF-β/Smad signaling pathway probably by upregulating Smad 7 expression and, thus, suppressing the expression of type I and type III collagens and TGF-β1.

150 PROTEOME OF BOVINE CUMULUS CELLS AS RELATED TO OOCYTE MORPHOLOGY AND IN VITRO EMBRYO PRODUCTION

2017 ◽  
Vol 29 (1) ◽  
pp. 183
Author(s):  
I. C. Velez ◽  
M. M. Ramirez ◽  
A. I. Chica ◽  
R. Urrego ◽  
A. A. Moura ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Silico ◽  
Triosephosphate Isomerase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Type I ◽  
Type Ii

The present study was conducted to study the effect of cumulus-oocyte complex (COC) morphology on subsequent in vitro embryo development and to assess the proteome of their corresponding cumulus cells (CC). Cow ovaries were obtained at an abattoir and COC aspirated from 3–8 mm follicles. The COC were defined as type I (TI): homogeneous ooplasma and ≥4 layers of compact CC; type II (TII): granular ooplasma and ≥4 layers of slight expanded CC. Fifty COC had ~500,000 CC. Cumulus cells were frozen in ammonium bicarbonate and immediately lyophilized for proteome analysis. Other selected COC were matured in vitro in TCM-199-supplemented media for 24 h. After maturation, CC were collected (T24) and processed as described above. The remaining COC were fertilized with sperm from a fertile bull and zygotes, cultured in vitro until Day 7. Ten blastocysts per group were stained (Hoechst 33342) and blastomeres, counted for assessment of embryo quality. The CC proteins were obtained from the following groups: immature type I (TIT0) and type II (TIIT0), and in vitro matured type I (TIT24) and type II (TIIT24). For protein extraction, we used sonication (30 min, 4°C), freezing and unfreezing in liquid nitrogen, and maceration. The CC proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by ESI-MS/MS. Differences in cleavage, embryo rates, and blastomere numbers were analysed by t-test. Protein expression difference was set at 2.5-fold (P < 0.05). In silico protein interactions were investigated using STRING v. 10.0. There were no differences in cleavage (88 ± 4 v. 89 ± 8%) and embryo rates (36 ± 7 v. 33 ± 8%) between COC of TI (n = 220) and TII (n = 161), respectively. Blastomeres were also similar in TI (101) and TII (104) groups. Major proteins expressed in all CC were α-enolase, β-actin, oestradiol 17-β-dehydrogenase 1, glutathione S-transferase, glyceraldehyde 3-phosphate dehydrogenase, heat shock protein β-1, histone H2B type 1-N, histone H4, mitochondrial malate dehydrogenase 2, protein disulfide isomarase A6, triosephosphate isomerase, tubulin α-1C chain, and vimentin. Glyceraldehyde 3-phosphate dehydrogenase appeared to be more expressed in TIT0, whereas tubulin α-1C chain and vimentin had greater expression in TIIT24. As evidenced by in silico analysis, most CC proteins interact among themselves, participating in complex networks involving intracellular signalling and other events. In conclusion, there are no difference in embryo development when using compact and early-expanded COC, indicating that both types can be selected for IVP. Protein profile of cumulus cell may serve as a marker for in vitro embryo competence.

scholarly journals Epigallocatechin-3-Gallate Promotes Osteo-/Odontogenic Differentiation of Stem Cells from the Apical Papilla through Activating the BMP–Smad Signaling Pathway

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1580
Author(s):  
Zeni Liu ◽  
Yuxiu Lin ◽  
Xiaolin Fang ◽  
Jingwen Yang ◽  
Zhi Chen
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Signaling Pathway ◽  
Matrix Protein ◽  
Type I ◽  
Alizarin Red ◽  
Smad Signaling ◽  
Odontogenic Differentiation ◽  
Smad Signaling Pathway ◽  
Apical Papilla

Stem cells from apical papilla (SCAPs) are desirable sources of dentin regeneration. Epigallocatechin-3-gallate (EGCG), a natural component of green tea, shows potential in promoting the osteogenic differentiation of bone mesenchymal stem cells. However, whether EGCG regulates the odontogenic differentiation of SCAPs and how this occurs remain unknown. SCAPs from immature human third molars (16–20 years, n = 5) were treated with a medium containing different concentrations of EGCG or bone morphogenic protein 2 (BMP2), with or without LDN193189 (an inhibitor of the canonical BMP pathway). Cell proliferation and migration were analyzed using a CCK-8 assay and wound-healing assay, respectively. Osteo-/odontogenic differentiation was evaluated via alkaline phosphatase staining, alizarin red S staining, and the expression of osteo-/odontogenic markers using qPCR and Western blotting. We found that EGCG (1 or 10 μM) promoted the proliferation of SCAPs, increased alkaline phosphatase activity and mineral deposition, and upregulated the expression of osteo-/odontogenic markers including dentin sialophosphoprotein (Dspp), dentin matrix protein-1 (Dmp-1), bone sialoprotein (Bsp), and Type I collagen (Col1), along with the elevated expression of BMP2 and phosphorylation level of Smad1/5/9 (p < 0.01). EGCG at concentrations below 10 μM had no significant influence on cell migration. Moreover, EGCG-induced osteo-/odontogenic differentiation was significantly attenuated via LDN193189 treatment (p < 0.01). Furthermore, EGCG showed the ability to promote mineralization comparable with that of recombinant BMP2. Our study demonstrated that EGCG promotes the osteo-/odontogenic differentiation of SCAPs through the BMP–Smad signaling pathway.

scholarly journals Pirfenidone Alleviates Choroidal Neovascular Fibrosis through TGF-β/Smad Signaling Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Chuang Gao ◽  
Xin Cao ◽  
Lili Huang ◽  
Yueqi Bao ◽  
Tao Li ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Transforming Growth Factor Β ◽  
Age Related Macular Degeneration ◽  
Type I ◽  
Fundus Fluorescein Angiography ◽  
Smad Signaling ◽  
Age Related ◽  
Smad Signaling Pathway

Background. Transforming growth factor-β (TGF-β) plays a major role in CNV. However, the mechanism is unclear. This study investigates the effect of Pirfenidone (PFD) on TGF-β/Smad signaling pathway on the development of choroidal neovascular fibrosis in choroidal neovascularization (CNV) mouse model. C57BL/6J male mice (aged from 6 to 8 weeks) received intravitreal injections of phosphate-buffered saline (PBS)/PFD solution on 14 days after laser injury. Mice were anesthetized by intraperitoneal injection of 4% pentobarbital (0.05 mg/g body weight). Optical Coherence Tomography (OCT), Fundus Fluorescein angiography (FFA), and hematoxylin-eosin (HE) were used to assess CNV formation. The fibrosis area was monitored by staining the collagen type I (Col-I). Western blotting was used to analyze the expression of TGF-β2, Smad 2/3, phosphorylated Smad 2/3 (p-Smad 2/3), and α-smooth muscle actin (α-SMA). Terminal deoxynucleotidy1 transferase dUTP nick-end labelling (TUNEL) assay was performed on cryosections of mouse eyes to detect apoptosis. Our data showed PFD inhibited areas of fibrosis during day 21 to day 28. We also found that the levels of TGF-β2 protein expressions increasingly reached the peak till the 3rd week during the CNV development. The protein levels of Smad 2/3, p-Smad 2/3, and α-SMA also increased significantly in CNV mice, but this response was profoundly suppressed by the TGF-β inhibitor PFD. The results of this study suggest that TGF-β2 represents a target to prevent or treat choroidal neovascular fibrosis, and PFD may provide an alternative to traditional methods for Wet Age-related macular degeneration (wAMD) treatment.

Inhibitory Effect of Corilagin on miR-21-Regulated Hepatic Fibrosis Signaling Pathway

2019 ◽  
Vol 47 (07) ◽  
pp. 1541-1569 ◽  
Author(s):  
Xuan Zhou ◽  
Jun Xiong ◽  
Shi Lu ◽  
Lei Luo ◽  
Zhi-Lin Chen ◽  
...  
Keyword(s):  
Growth Factor ◽  
Liver Fibrosis ◽  
Signaling Pathway ◽  
Hepatic Fibrosis ◽  
Type I ◽  
Loss Of Function ◽  
Smad Signaling ◽  
Protein Levels ◽  
Smad Signaling Pathway ◽  
Liver Tissues

Corilagin is a polyphenol that can be extracted from many medicinal plants and shows multiple pharmacological effects. We aimed to investigate the role of corilagin on miR-21-regulated hepatic fibrosis, especially miR-21-regulated TGF-[Formula: see text]1/Smad signaling pathway, in hepatic stellate LX2 cell line and Sprague–Dawley rats. The mRNA or protein levels of miR-21, Smad7, connective tissue growth factor (CTGF), [Formula: see text]-smooth muscle actin ([Formula: see text]-SMA), tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), collagen type I alpha 1 (COL1A1), Smad2, Smad3, Smad2/3, p-Smad2, p-Smad3, p-Smad2/3, and transforming growth factor-[Formula: see text]1 (TGF-[Formula: see text]1) in LX2 cells and liver tissues were determined. Furthermore, gain-of and loss-of function of miR-21 in miR-21-regulated TGF-[Formula: see text]1/Smad signaling pathway were analyzed in LX2 cells. Liver tissues and serum were collected for pathological analysis, immunohistochemical staining, and enzyme-linked immunosorbent assay (ELISA). Corilagin treatment reduced mRNA or protein levels of miR-21, CTGF, [Formula: see text]-SMA, TIMP-1, TGF-[Formula: see text]1, COL1A1, p-Smad2, p-Smad3, and p-Smad2/3 both in vitro and in vivo. While corilagin increased mRNA and protein levels of Smad7 and MMP-9. After gain-of and loss-of function of miR-21, the downstream effectors of miR-21-regulated TGF-[Formula: see text]1/Smad signaling pathway in LX2 cells changed accordingly, and the changes were inhibited by corilagin. Simultaneously, administration of corilagin not only ameliorated pathological manifestation of liver fibrosis but also reduced levels of [Formula: see text]-SMA and COL1A1 in liver tissues and TGF-[Formula: see text]1, ALT levels in serum. Corilagin is able to potentially prevent liver fibrosis by blocking the miR-21-regulated TGF-[Formula: see text]1/Smad signaling pathway in LX2 cells and CCl4-induced liver fibrosis rats, which may provide a novel therapeutic strategy for liver fibrosis.

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