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BACKGROUND: Bone marrow mesenchymal stem cells (BMMSCs) are commonly used for cell transplantation to treat refractory diseases. However, the presence of inflammatory factors, such as tumour necrosis factor-alpha (TNF-α), at the transplantation site severely compromises the stemness of BMMSCs, thereby reducing the therapeutic effect of cell transplantation. Aspirin (AS) is a drug that has been in use for over a century and has a wide range of effects, including the regulation of cell proliferation, multidirectional differentiation, and immunomodulatory properties of stem cells. However, it is still unclear whether AS can delay the damaging effects of TNF-α on BMMSC stemness. METHODS: This study investigated the effects of AS and TNF-α on BMMSC stemness and the molecular mechanisms using colony formation assay, western blot, qRT-PCR, and overexpression or knockdown of YAP and SMAD7. RESULTS: The results demonstrated that TNF-α inhibited cell proliferation, the expression of stemness, osteogenic and chondrogenic differentiation markers of BMMSCs. Treatment with AS was shown to mitigate the TNF-α-induced damage to BMMSC stemness. Mechanistic studies revealed that AS may reverse the damage caused by TNF-α on BMMSC stemness by upregulating YAP and inhibiting the expression of SMAD7. CONCLUSION: AS can attenuate the damaging effects of TNF-α on BMMSC stemness by regulating the YAP-SMAD7 axis. These findings are expected to promote the application of AS to improve the efficacy of stem cell therapy.
Subject(s)
Aspirin , Cell Differentiation , Cell Proliferation , Mesenchymal Stem Cells , Smad7 Protein , Tumor Necrosis Factor-alpha , YAP-Signaling Proteins , Tumor Necrosis Factor-alpha/metabolism , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/drug effects , Smad7 Protein/metabolism , Smad7 Protein/genetics , Aspirin/pharmacology , Cell Proliferation/drug effects , Cell Differentiation/drug effects , YAP-Signaling Proteins/metabolism , Signal Transduction/drug effects , Animals , Transcription Factors/metabolism , Transcription Factors/genetics , Humans , Cells, Cultured , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Osteogenesis/drug effects , MiceABSTRACT
BACKGROUND: Hepatic fibrosis (HF) is an essential stage in the progression of different chronic liver conditions to cirrhosis and even hepatocellular carcinoma. The activation of hepatic stellate cells (HSCs) plays a crucial role in the progression of HF. IFN- γ/Smad7 pathway can inhibit HSCs activation, while TGF-ß1/CUGBP1 pathway can inhibit IFN-γ/Smad7 pathway transduction and promote HSCs activation. Thus, inhibiting the TGF-ß1/CUGBP1 pathway and activating the IFN-γ/Smad7 pathway reverses HSCs activation and inhibits HF. Jiawei Taohe Chengqi Decoction (JTCD) was derived from the Taohe Chengqi Tang in the ancient Chinese medical text titled "Treatise on Febrile Diseases". We found several anti-HF components in JTCD including ginsenoside Rb1 and others, but the specific mechanism of anti-HF in JTCD is not clear. PURPOSE: To elucidate the specific mechanism by which JTCD reverses HF by inhibiting the activation of HSCs, and to establish a scientific foundation for treating HF with Traditional Chinese medicine (TCM). METHODS: We constructed a CCl4-induced mice HF model in vivo and activated human hepatic stellate cell line (LX-2) with TGF-ß1 in vitro, after which they were treated with JTCD and the corresponding inhibitors. We examined the expression of pivotal molecules in the two pathways mentioned above by immunofluorescence staining, Western blotting and RT-PCR. RESULTS: JTCD attenuated liver injury and reduced serum ALT and AST levels in mice. In addition, JTCD attenuated CCl4-induced HF by decreasing the expression of α-SMA, COL1A1 and other markers of HSCs activation in mice liver tissue. Moreover, JTCD effectively suppressed the levels of TGF-ß1, p-Smad3, p-p38MAPK, p-ATF2, and CUGBP1 in vivo and in vitro and upregulated the levels of IFN-γ, p-STAT1, and Smad7. Mechanically, after using the inhibitors of both pathways in vitro, we found that JTCD inhibited the activation of HSCs by restoring the balance of the TGF-ß1/CUGBP1 and IFN-γ/Smad7 pathways. CONCLUSION: We demonstrated that JTCD inhibited HSCs activation and reversed HF by inhibiting the TGF-ß1/CUGBP1 signalling pathway and upregulating the IFN-γ/Smad7 signalling pathway. Moreover, we have identified specific links where JTCD interferes with both pathways to inhibit HSCs activation. JTCD is an effective candidate for the clinical treatment of HF.
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OBJECTIVE: To examine the influence of Saponin I from Shuitianqi (Rhizoma Schizocapasae Plantagineae) (SSPH I) on hepatocellular carcinoma (HCC) metastasis, and to elucidate the underlying mechanism. METHODS: The intrahepatic metastasis Bagg's Albino/c (BALB/c) mouse model was established with human hepatocellular carcinomas (HepG2) cells, then treated with normal saline (once per day), cisplatin (2 mg/kg, once every 2 d), and SSPH â (25, 50, and 75 mg/kg, once per day). Then, we assessed alterations in the hepatic pathology and target protein expressions in the intrahepatic metastasis BALB/c mouse model using a series of molecular biology techniques. RESULTS: Based on our analysis, SSPH â significantly alleviated hepatocyte necrosis and tumor cells infiltration. Moreover, SSPH â suppressed extracellular matrix (ECM) degradation and angiogenesis viaa decrease in matrix etalloproteinase-2 (MMP-2), MMP-9, CD31, CD34, and vascular endothelial growth factor (VEGF) levels. Furthermore, SSPH â repressed invasion and meta-stasis by suppressing the transforming growth factor-ß1 (TGF-ß1)/Smad7 axis and epithelial-mesenchymal transition (EMT), as evidenced by the scarce TGF-ß1, N-cadherin, and Vimentin expressions, and elevated Smad7 and E-cadherin expressions. CONCLUSION: The SSPH â -mediated negative regulation of the TGF-ß1/Smad7 axis and EMT are critical for the inhibition of HCC invasion and metastasis.
Subject(s)
Drugs, Chinese Herbal , Epithelial-Mesenchymal Transition , Liver Neoplasms , Saponins , Smad7 Protein , Transforming Growth Factor beta1 , Animals , Humans , Male , Mice , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/genetics , Disease Models, Animal , Drugs, Chinese Herbal/administration & dosage , Drugs, Chinese Herbal/pharmacology , Epithelial-Mesenchymal Transition/drug effects , Hep G2 Cells , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Liver Neoplasms/genetics , Neoplasm Metastasis , Saponins/pharmacology , Smad7 Protein/metabolism , Smad7 Protein/genetics , Transforming Growth Factor beta1/metabolism , Transforming Growth Factor beta1/geneticsABSTRACT
Long-term denervation-induced atrophy and fibrosis of skeletal muscle due to denervation leads to poor recovery of muscle function. Studies have shown that the transforming growth factor-ß1 (TGF-ß1)-Smad signaling pathway plays a central role in muscle atrophy and fibrosis. Recent studies demonstrate the role of microRNAs (miRs) in various pathological conditions, including muscle regeneration. miR-21 has been shown to play a dynamic role in inflammatory responses and in accelerating injury responses to fibrosis. We used both RNA sequencing and quantitative RT-PCR strategies to examine the alternations of miRNAs during denervation-induced gastrocnemius muscle atrophy and fibrosis. Our data showed that MiR-21 was upregulated in denervated gastrocnemius muscle tissue, and TGF-ß1treatment increased miR-21 expression. Inhibition of miR-21 reduced gastrocnemius muscle fibrosis and significantly downregulated the expression of p-SMAD2/3 and the fibrosis-associated markers TGF-ß1, connective tissue growth factor, alpha smooth muscle actin. Masson's trichrome staining revealed that atrophy and fibrosis in gastrocnemius muscle tissue were reduced in the miR-21 inhibition group compared to the control group. We confirmed that SMAD7 is a direct target of miR-21 using a dual luciferase assay. Furthermore, Immunofluorescence and Western blot analyses revealed that miR-21 inhibition reduced SMAD2/3 phosphorylation and nuclear translocation. While SMAD7-siRNA abolished the effect. Consequently, the discovery that miR-21 regulates the atrophy and fibrosis of the gastrocnemius muscle offers a possible therapeutic approach for their management.
ABSTRACT
The reorganization of the cell cytoskeleton and changes in the content of cell adhesion molecules are crucial during the metastatic spread of tumor cells. Colorectal cancer (CRC) cells express high SMAD7, a protein involved in the control of CRC cell growth. In the present study, we evaluated whether SMAD7 regulates the cytoskeleton reorganization and dynamics in CRC. Knockdown of SMAD7 with a specific antisense oligonucleotide (AS) in HCT116 and DLD1, two human CRC cell lines, reduced the migration rate and the content of F-ACTIN filaments. A gene array, real-time PCR, and Western blotting of SMAD7 AS-treated cells showed a marked down-regulation of the X-linked inhibitor of apoptosis protein (XIAP), a member of the inhibitor of apoptosis family, which has been implicated in cancer cell migration. IL-6 and IL-22, two cytokines that activate STAT3, enhanced XIAP in cancer cells, and such induction was attenuated in SMAD7-deficient cells. Finally, in human CRC, SMAD7 mRNA correlated with XIAP expression. Our data show that SMAD7 positively regulates XIAP expression and migration of CRC cells, and suggest a mechanism by which SMAD7 controls the architecture components of the CRC cell cytoskeleton.
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BACKGROUND: The previous study has proved the oncogenic role of Yes-associated protein 1 (YAP1) in bladder cancer (BLCA), thus this study focused on its impact on bladder cancer stem cells (BCSCs) and underlying mechanism. METHOD: BCSCs were obtained by treating human BLCA cells UMUC3 with cisplatin and identified by measuring CD133+ in UMUC3/BCSCs via flow cytometry. YAP1 interaction proteins and mothers against decapentaplegic homolog 7 (SMAD7) N6-methyladenosine (m6A) site were analyzed by bioinformatics. BCSCs were transfected. SMAD7 m6A level, YTH domain-containing family proteins 3 (YTHDF3)-SMAD7 interaction, YAP1/YTHDF3 expression in BCSCs were assessed by methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP) or quantitative reverse transcription PCR (qRT-PCR), respectively. BCSC proliferation was detected by 5-Bromo-2-deoxyuridine (BrdU) staining. UMUC3/BCSC migration/invasion and tumour sphere formation were determined by Transwell or tumour sphere formation assays. YAP1/YTHDF3/SMAD7/transforming growth factor (TGF)-ß1/stemness marker expressions in UMUC3/BCSCs were measured by Western blot assay. RESULT: BCSCs showed higher CD133+ ratio, expressions of stemness marker/YAP1/YTHDF3/TGF-ß1, lower SMAD7 expression and greater invasion/migration/tumour sphere formation capabilities than UMUC3 cells. YAP1 knockdown decreased SMAD7 m6A level and impaired YTHDF3-SMAD7 interaction in BCSCs. YAP1 silencing inhibited cell growth/invasiveness/migration/tumour sphere formation and stemness-associated protein/YTHDF3/TGF-ß1 expressions while upregulating SMAD7 expression in BCSCs, which was offset by YTHDF3 overexpression. CONCLUSION: The silencing of YAP1 in BCSCs impedes the YTHDF3-mediated degradation of m6A-modified SMAD7, culminating in diminished cell stemness.
Subject(s)
Adaptor Proteins, Signal Transducing , Neoplastic Stem Cells , RNA-Binding Proteins , Smad7 Protein , Transcription Factors , Urinary Bladder Neoplasms , YAP-Signaling Proteins , Humans , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adenosine/analogs & derivatives , Adenosine/metabolism , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Smad7 Protein/metabolism , Smad7 Protein/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/metabolism , YAP-Signaling Proteins/metabolism , YAP-Signaling Proteins/geneticsABSTRACT
BACKGROUND: Enhanced glycolysis is a crucial metabolic event that drives the development of liver fibrosis, but the molecular mechanisms have not been fully understood. Lactate is the endproduct of glycolysis, which has recently been identified as a bioactive metabolite binding to G-protein-coupled receptor 81 (GPR81). We then questioned whether GPR81 is implicated in the development of liver fibrosis. METHODS: The level of GPR81 was determined in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and in transforming growth factor beta 1 (TGF-ß1)-activated hepatic stellate cells (HSCs) LX-2. To investigate the significance of GPR81 in liver fibrosis, wild-type (WT) and GPR81 knockout (KO) mice were exposed to CCl4, and then the degree of liver fibrosis was determined. In addition, the GPR81 agonist 3,5-dihydroxybenzoic acid (DHBA) was supplemented in CCl4-challenged mice and TGF-ß1-activated LX-2 cells to further investigate the pathological roles of GPR81 on HSCs activation. RESULTS: CCl4 exposure or TGF-ß1 stimulation significantly upregulated the expression of GPR81, while deletion of GPR81 alleviated CCl4-induced elevation of aminotransferase, production of pro-inflammatory cytokines, and deposition of collagen. Consistently, the production of TGF-ß1, the expression of alpha-smooth muscle actin (α-SMA) and collagen I (COL1A1), as well as the elevation of hydroxyproline were suppressed in GPR81 deficient mice. Supplementation with DHBA enhanced CCl4-induced liver fibrogenesis in WT mice but not in GPR81 KO mice. DHBA also promoted TGF-ß1-induced LX-2 activation. Mechanistically, GPR81 suppressed cAMP/CREB and then inhibited the expression of Smad7, a negative regulator of Smad3, which resulted in increased phosphorylation of Smad3 and enhanced activation of HSCs. CONCLUSION: GPR81 might be a detrimental factor that promotes the development of liver fibrosis by regulating CREB/Smad7 pathway.
Subject(s)
Carbon Tetrachloride , Cyclic AMP Response Element-Binding Protein , Hepatic Stellate Cells , Liver Cirrhosis , Mice, Knockout , Receptors, G-Protein-Coupled , Signal Transduction , Smad7 Protein , Animals , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/genetics , Liver Cirrhosis/metabolism , Liver Cirrhosis/genetics , Liver Cirrhosis/pathology , Liver Cirrhosis/etiology , Liver Cirrhosis/chemically induced , Mice , Cyclic AMP Response Element-Binding Protein/metabolism , Hepatic Stellate Cells/metabolism , Smad7 Protein/metabolism , Smad7 Protein/genetics , Transforming Growth Factor beta1/metabolism , Male , Humans , Cell Line , Disease Models, Animal , Mice, Inbred C57BL , Gene DeletionABSTRACT
Intracellular signaling by the pleiotropic cytokine transforming growth factor-ß (TGF-ß) is inhibited by Smad7 in a feedback control mechanism. The activity of Smad7 is tightly regulated by multiple post-translational modifications. Using resin-assisted capture and metabolic labeling methods, we show here that Smad7 is S-palmitoylated in mammary epithelial cell models that are widely studied because of their strong responses to TGF-ß and their biological relevance to mammary development and tumor progression. S-palmitoylation of Smad7 is mediated by zDHHC17, a member of a family of 23 S-acyltransferase enzymes. Moreover, we identified four cysteine residues (Cys202, Cys225, Cys415, and Cys417) in Smad7 as palmitoylation acceptor sites. S-palmitoylation of Smad7 on Cys415 and Cys417 promoted the translocation of Smad7 from the nucleus to the cytoplasm, enhanced the stability of the Smad7 protein, and enforced its inhibitory effect on TGF-ß-induced Smad transcriptional response. Thus, our findings reveal a new post-translational modification of Smad7, and highlight an important role of S-palmitoylation to enhance inhibition of TGF-ß/Smad signaling by Smad7.
Subject(s)
Acyltransferases , Lipoylation , Signal Transduction , Smad7 Protein , Transforming Growth Factor beta , Smad7 Protein/metabolism , Smad7 Protein/genetics , Humans , Acyltransferases/metabolism , Acyltransferases/genetics , Transforming Growth Factor beta/metabolism , HEK293 Cells , Protein Processing, Post-Translational , Animals , Cell Nucleus/metabolism , Cysteine/metabolismABSTRACT
Dystrophic cardiomyopathy is a significant feature of Duchenne muscular dystrophy (DMD). Increased cardiomyocyte cytosolic calcium (Ca2+) and interstitial fibrosis are major pathophysiological hallmarks that ultimately result in cardiac dysfunction. MicroRNA-25 (miR-25) has been identified as a suppressor of both sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and mothers against decapentaplegic homolog-7 (Smad7) proteins. In this study, we created a gene transfer using an miR-25 tough decoy (TuD) RNA inhibitor delivered via recombinant adeno-associated virus serotype 9 (AAV9) to evaluate the effect of miR-25 inhibition on cardiac and skeletal muscle function in aged dystrophin/utrophin haploinsufficient mice mdx/utrn (+/-), a validated transgenic murine model of DMD. We found that the intravenous delivery of AAV9 miR-25 TuD resulted in strong and stable inhibition of cardiac miR-25 levels, together with the restoration of SERCA2a and Smad7 expression. This was associated with the amelioration of cardiomyocyte interstitial fibrosis as well as recovered cardiac function. Furthermore, the direct quadricep intramuscular injection of AAV9 miR-25 TuD significantly restored skeletal muscle Smad7 expression, reduced tissue fibrosis, and enhanced skeletal muscle performance in mdx/utrn (+/-) mice. These results imply that miR-25 TuD gene transfer may be a novel therapeutic approach to restore cardiomyocyte Ca2+ homeostasis and abrogate tissue fibrosis in DMD.
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In this study, we sought to investigate the mechanisms of action of miR-195-5p in the osteogenic differentiation of vascular smooth muscle cells (VSMCs), and thereby provide novel insights and a reference for the targeted therapy of arterial media calcification. VSMC differentiation was induced using sodium ß-glycerophosphate, and we investigated the effects of transfecting cells with miR-195-5p mimics, vectors overexpressing Smad7, and the Wnt/ß-catenin pathway inhibitor (KYA1797K) on VSMC differentiation by determining cell viability and apoptosis, and the mRNA and protein expression of factors associated with osteogenic differentiation and the Wnt/ß-catenin pathway. The results revealed that miR-195-5p mimics enhanced the osteogenic differentiation of VSMCs induced by ß-glycerophosphate, whereas the overexpression of Smad7 reversed this phenomenon. In addition, KYA1797K was found to promote the effects of Smad7 overexpression. In conclusion, by targeting, Smad7, miR-195-5p promotes the Wnt/ß-catenin pathway. and thus the osteogenic differentiation of VSMCs. These findings will provide a reference for elucidating the mechanisms whereby miR-195-5p regulates osteogenic differentiation.
Subject(s)
Cell Differentiation , MicroRNAs , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , Osteogenesis , Smad7 Protein , Wnt Signaling Pathway , Animals , Apoptosis , beta Catenin/metabolism , beta Catenin/genetics , Cells, Cultured , Gene Expression Regulation , Glycerophosphates/pharmacology , MicroRNAs/genetics , MicroRNAs/metabolism , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/drug effects , Osteogenesis/genetics , Smad7 Protein/metabolism , Smad7 Protein/genetics , RatsABSTRACT
BACKGROUND: Endothelial cell injury and dysfunction lead to cholesterol and lipid accumulation and atherosclerotic plaque formation in the arterial wall during atherosclerosis (AS) progression, Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), a DNA methylation regulator, was strongly upregulated in atherosclerotic plaque lesions in mice. This study aimed to investigate the precise biological functions and regulatory mechanisms of UHRF1 on endothelial dysfunction during AS development. METHODS: UHRF1 levels in the atherosclerotic plaque tissues and normal arterial intima from AS patients were tested with Western blot analysis and immunohistochemistry assays. Human umbilical vein endothelial cells (HUVECs) were stimulated with oxidized low-density lipoprotein (ox-LDL) to induce an injury model and then transfected with short hairpin RNA targeting UHRF1 (sh-UHRF1). Cell proliferation, migration, apoptosis, the levels of inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and the protein levels adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were measured. Moreover, co-immunoprecipitation assay was used to determine the interactions between UHRF1 and DNA methyltransferases 1 (DNMT1), As well as mothers against DPP homolog 7 (SMAD7) and yes-associated protein 1 (YAP1). SMAD7 promoter methylation was examined with methylation-specific PCR. In addition, we established an AS mouse model to determine the in vivo effects of UHRF1 on AS progression. RESULTS: UHRF1 was upregulated in atherosclerotic plaque tissues and ox-LDL-treated HUVECs. UHRF1 knockdown mitigated ox-LDL-induced proliferation and migration inhibition, apoptosis and the production of TNF-α, IL-6, VCAM-1, and ICAM-1 in HUVECs. Mechanistically, UHRF1 promoted DNMT1-mediated SMAD7 promoter methylation and inhibited its expression. SMAD7 knockdown abolished the protective effects of UHRF1 knockdown on ox-LDL-induced HUVEC injury. Moreover, SMAD7 interacted with YAP1 and inhibited YAP1 expression by promoting YAP1 protein ubiquitination-independent degradation in HUVECs. YAP1 overexpression abrogated SMAD7 overexpression-mediated protective effects on ox-LDL-induced HUVEC injury. Finally, UHRF1 knockdown alleviated atherosclerotic plaque deposition and arterial lesions in AS mice. CONCLUSION: UHRF1 inhibition mitigates vascular endothelial cell injury and ameliorates AS progression in mice by regulating the SMAD7/YAP1 axis.
Subject(s)
Atherosclerosis , Smad7 Protein , Ubiquitin-Protein Ligases , YAP-Signaling Proteins , Animals , Humans , Male , Mice , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis/drug effects , Atherosclerosis/metabolism , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Proliferation , Human Umbilical Vein Endothelial Cells , Lipoproteins, LDL/metabolism , Mice, Inbred C57BL , Plaque, Atherosclerotic/metabolism , Plaque, Atherosclerotic/pathology , Signal Transduction , Smad7 Protein/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , YAP-Signaling Proteins/metabolismABSTRACT
Appropriate fibrosis is required to prevent subsequent adverse remodeling and heart failure post myocardial infarction (MI), and cardiac fibroblasts (CFs) play a critical role during the process. Carbonic anhydrase 3 (CAR3) is an important mediator in multiple biological processes besides its CO2 hydration activity; however, the role and underlying mechanism of CAR3 on cardiac repair post MI injury remains unknown. Here, we found that CAR3 expression was up-regulated in cardiac tissue in infarct area at the reparative phase of MI, with a peak at 7 days post MI. The upregulation was detected mainly on fibroblast instead of cardiomyocyte, and primary cardiac fibroblasts treated with TGF-ß1 recaptured our observation. While CAR3 deficiency leads to weakened collagen density, enlarged infarct size and aggravated cardiac dysfunction post-MI. In fibroblast, we observed that CAR3 deficiency restrains collagen synthesis, cell migration and gel contraction of cardiac fibroblasts, whereas overexpression of CAR3 in CFs improves wound healing and cardiac fibroblast activation. Mechanistically, CAR3 stabilizes Smad7 protein via modulating its acetylation, which dampens phosphorylation of Smad2 and Smad3, thus inhibiting fibroblast transformation. In contrast, inhibition of Smad7 acetylation with C646 blunts CAR3 deficiency induced suppression of fibroblast activation and impaired cardiac healing. Our data demonstrate a protective role of CAR3 in cardiac wound repair post MI via promoting fibroblasts activation through Smad7-TGF-ß/Smad2/3 signaling pathway.
Subject(s)
Carbonic Anhydrases , Myocardial Infarction , Humans , Myocardium/metabolism , Smad7 Protein/metabolism , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , Signal Transduction/genetics , Myocytes, Cardiac/metabolism , Transforming Growth Factor beta1/metabolism , Collagen/metabolism , Carbonic Anhydrases/metabolism , Fibroblasts/metabolismABSTRACT
The capillarization of hepatic sinusoids resulting from the activation of hepatic stellate cells poses a significant challenge, impeding the effective delivery of therapeutic agents to the Disse space for liver fibrosis treatment. Therefore, overcoming these barriers and achieving efficient drug delivery to activated hepatic stellate cells (aHSCs) are pressing challenge. In this study, we developed a synergistic sequential drug delivery approach utilizing neutrophil membrane hybrid liposome@atorvastatin/amlisentan (NCM@AtAm) and vitamin A-neutrophil membrane hybrid liposome @albumin (VNCM@Bai) nanoparticles (NPs) to breach the capillary barrier for targeted HSC cell delivery. Initially, NCM@AtAm NPs were successfully directed to the site of hepatic fibrosis through neutrophil-mediated inflammatory targeting, resulting in the normalization of liver sinusoidal endothelial cells (LSECs) and restoration of fenestrations under the combined influence of At and Am. Elevated tissue levels of the p-Akt protein and endothelial nitric oxide synthase (eNOS) indicated the normalization of LSECs following treatment with At and Am. Subsequently, VNCM@Bai NPs traversed the restored LSEC fenestrations to access the Disse space, facilitating the delivery of Bai into aHSCs under vitamin A guidance. Lastly, both in vitro and in vivo results demonstrated the efficacy of Bai in inhibiting HSC cell activation by modulating the PPAR γ/TGF-ß1 and STAT1/Smad7 signaling pathways, thereby effectively treating liver fibrosis. Overall, our designed synergistic sequential delivery system effectively overcomes the barrier imposed by LSECs, offering a promising therapeutic strategy for liver fibrosis treatment in clinical settings.
Subject(s)
Endothelial Cells , Hepatic Stellate Cells , Humans , Endothelial Cells/metabolism , Bionics , Capillaries/metabolism , Liposomes/metabolism , Neutrophils/metabolism , Vitamin A/metabolism , Vitamin A/pharmacology , Liver/metabolism , Liver Cirrhosis/drug therapy , Liver Cirrhosis/metabolismABSTRACT
INTRODUCTION: The NLR family pyrin domain containing 3 (NLRP3)-mediated pyroptosis was positively correlated with the allergic rhinitis progression and was reported to be regulated by SMAD family member 7 (Smad7). Bioinformatics analysis revealed that Smad7 might be targeted by miR-96-5p, and miR-96-5p might be targeted by long noncoding RNA zinc finger antisense 1 (ZFAS1). However, the effects and regulatory mechanisms of the ZFAS1/miR-96-5p/Smad7 functional axis in allergic rhinitis have not been investigated. METHODS: Human nasal mucosa epithelial cell line RPMI 2650 and C57BL/6 mice were obtained for in vitro and in vivo studies. Dual-luciferase reporter assay and RNA immunoprecipitation were implemented for detecting molecular interactions. Cell counting kit-8 and flow cytometry were used for measuring cell viability and pyroptosis. ELISA was obtained for monitoring cytokine secretion. RT-qPCR and Western blot were examined for determining RNA and protein expression. RESULTS: In vitro studies revealed that ZFAS1 was downregulated in interleukin (IL)-13-treated RPMI 2650 cells, while overexpression of ZFAS1 enhanced cell viability and inhibited NLRP3-mediated pyroptosis and inflammatory response. ZFAS1 directly inhibited miR-96-5p to suppress NLRP3-mediated pyroptosis in IL-13-treated RPMI 2650 cells. MiR-96-5p bound to the 3'-untranslated region of Smad7 and knockdown of Smad7 significantly reversed the effects of miR-96-5p depletion. Moreover, in vivo experiments further confirmed the findings of in vitro studies and showed ZFAS1 overexpression or miR-96-5p inhibition alleviated allergic rhinitis in vivo. CONCLUSION: ZFAS1 downregulated the expression of miR-96-5p to upregulate Smad7 level, which subsequently inhibited NLRP3-mediated pyroptosis and inflammatory response to ameliorate allergic rhinitis.
Subject(s)
MicroRNAs , NLR Family, Pyrin Domain-Containing 3 Protein , Pyroptosis , RNA, Long Noncoding , Rhinitis, Allergic , Signal Transduction , Smad7 Protein , Animals , Humans , Mice , Cell Line , Disease Models, Animal , Inflammasomes/metabolism , Mice, Inbred C57BL , MicroRNAs/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Pyroptosis/genetics , Rhinitis, Allergic/metabolism , Rhinitis, Allergic/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Smad7 Protein/genetics , Smad7 Protein/metabolismABSTRACT
OBJECTIVE: 2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione (DMDD) isolated from Averrhoa carambola L. root, has been proven therapeutic effects on diabetic kidney disease (DKD). This research aims to assess DMDD's effects on DKD and to investigate its underlying mechanisms, to establish DMDD as a novel pharmaceutical agent for DKD treatment. METHODS: The human renal tubular epithelial (HK-2) cells were induced by high glucose (HG) to mimic DKD and followed by DMDD treatment. The cytotoxicity of DMDD was assessed using the Cell Counting Kit-8 (CCK-8) assay. The migratory capacity of HK-2 cells was evaluated through transwell and scratch-wound assays. To investigate the effect of Smad7 and miR-21-5p, lentiviral transfection was employed in HK-2 cells. Additionally, the expression of proteins related to epithelial-mesenchymal transition (EMT) and TGFß1/Smad2/3 pathway was checked by QRT-PCR, Western blot, and immunofluorescence techniques. RESULTS: This study has shown that DMDD significantly suppresses cell migration and the expression of Vimentin, α-SMA, TGFß1, and p-Smad2/3 in HK-2 cells under HG conditions. Concurrently, DMDD enhances the protein expression of E-cadherin and Smad7. Intriguingly, the therapeutic effect of DMDD was abrogated upon Smad7 silencing. Further investigations revealed that DMDD effectively inhibits miR-21-5p expression, which is upregulated by HG. Downregulation of miR-21-5p inhibits the activation of the TGFß1/Smad2/3 pathway and EMT induced by HG. In contrast, overexpression of miR-21-5p negates DMDD's therapeutic benefits. CONCLUSION: DMDD mitigates EMT in HG-induced HK-2 cells by modulating the miR-21-5p/Smad7 pathway, thereby inhibiting renal fibrosis in DKD. These findings suggest that DMDD holds promise as a potential therapeutic agent for DKD.
Subject(s)
Averrhoa , Cyclohexenes , Diabetic Nephropathies , Signal Transduction , Humans , Epithelial-Mesenchymal Transition , Glucose/metabolism , MicroRNAs/metabolism , Cyclohexenes/pharmacology , Diabetic Nephropathies/drug therapyABSTRACT
Activin A promotes the development of endometriotic lesions in a murine model of endometriosis, and the immunohistochemical localization of phosphorylated suppressor of mothers against decapentaplegic homolog 2/3 (pSMAD2/3) complex in endometriotic lesions has been reported. Activin may therefore be involved in the development and proliferation of endometriotic cells via the SMAD signaling pathway. However, few detailed reports exist on SMAD7 expression in endometriosis. The purpose of this study was to investigate the expression of pSMAD2/3 or pSMAD3 and SMAD7 in the orthotopic human endometrium, ovarian endometriosis, and endometriotic lesions in a murine model and the effect of activin A on pSMAD2/3 and SMAD7 expression. We established an endometriosis murine model via the intraperitoneal administration of endometrial tissue and blood from donor mice. Activin A was intraperitoneally administered to the activin group. We immunohistochemically evaluated orthotopic endometria, ovarian endometriotic tissues, and endometriotic lesions in the murine model followed by western blotting. We found that pSMAD3 and SMAD7 were expressed in ovarian endometriosis and orthotopic endometria from patients with and without endometriosis. In the murine model, endometriotic lesions expressed pSMAD2/3 and SMAD7 in the activin and control groups, and higher SMAD7 expression was found in the activin group. To the best of our knowledge, this study is the first to show that SMAD7 expression is upregulated in endometriosis. In conclusion, these results suggest that activin A activates the SMAD signaling pathway and promotes the development of endometriotic lesions, thus identifying SMAD7 as a potential therapeutic target for endometriosis.
Subject(s)
Activins , Disease Models, Animal , Endometriosis , Endometrium , Smad2 Protein , Smad3 Protein , Smad7 Protein , Endometriosis/metabolism , Endometriosis/pathology , Female , Animals , Humans , Endometrium/metabolism , Endometrium/pathology , Mice , Smad7 Protein/metabolism , Smad3 Protein/metabolism , Smad2 Protein/metabolism , Activins/metabolism , Ovarian Diseases/metabolism , Ovarian Diseases/pathology , Adult , Signal TransductionABSTRACT
Long intergenic non-coding RNA(lincRNA) is transcribed from the intermediate regions of coding genes and plays a pivotal role in the regulation of lipid synthesis. N6-methyladenosine (m6A) modification is widely prevalent in eukaryotic mRNAs and serves as a regulatory factor in diverse biological processes. This study aims to delineate the mechanism by which Linc-smad7 mediates m6A methylation to regulate milk fat synthesis. Tissue expression analysis in this study revealed a high expression of Linc-smad7 in breast tissue during pregnancy. Cell proliferation assays, including CCK8 and EdU assays, demonstrated that Linc-smad7 had no significant impact on the proliferation of mammary epithelial cells. However, during mammary epithelial cell differentiation, the overexpression of Linc-smad7 led to reduced lipid formation, whereas interference with Linc-smad7 promoted lipogenesis. Mechanistically, Linc-smad7 was found to modulate RNA m6A levels, as evidenced by dot blot assays and methylated RNA immunoprecipitation sequencing (MeRIP-Seq). Subsequent validation through RT-qPCR corroborated these findings, aligning with the m6A sequencing outcomes. Furthermore, co-transfection experiments elucidated that Linc-smad7 regulates lipid synthesis in mammary epithelial cells by influencing the expression of METTL14. In summary, these findings underscore the regulatory role of Linc-smad7 in controlling METTL14 gene expression, thereby mediating m6A modifications to regulate lipid synthesis in mammary epithelial cells.
Subject(s)
Epithelial Cells , Lipogenesis , RNA, Long Noncoding , Animals , Mice , Cell Differentiation , Lipids , Lipogenesis/genetics , RNA, Messenger , RNA, Long Noncoding/genetics , Smad7 Protein/genetics , RNA Methylation/genetics , Mammary Glands, Animal/cytology , Mammary Glands, Animal/metabolismABSTRACT
Atherosclerosis is one of the leading causes of death worldwide. miR-26 is a potential biomarker of atherosclerosis. Standardized diagnostic tests for miR-26 (MIR26-DX) have been developed, but the fastest progress has been in predicting the efficacy of IFN-α therapy for hepatocellular carcinoma (HCC, phase 3). MiR-26 slows atherosclerosis development by suppressing ACC1/2, ACLY, ACSL3/4, ALDH3A2, ALPL, BMP2, CD36, COL1A1, CPT1A, CTGF, DGAT2, EHHADH, FAS, FBP1, GATA4, GSK3ß, G6PC, Gys2, HMGA1, HMGB1, LDLR, LIPC, IL-1ß, IL-6, JAG2, KCNJ2, MALT1, ß-MHC, NF-κB, PCK1, PLCß1, PYGL, RUNX2, SCD1, SMAD1/4/5/7, SREBF1, TAB3, TAK1, TCF7L2, and TNF-α expression. Many agents targeting these genes, such as the ACC1/2 inhibitors GS-0976, PF-05221304, and MK-4074; the DGAT2 inhibitors IONIS-DGAT2Rx, PF-06427878, PF-0685571, and PF-07202954; the COL1A1 inhibitor HT-100; the stimulants 68Ga-CBP8 and RCT-01; the CPT1A inhibitors etomoxir, perhexiline, and teglicar; the FBP1 inhibitors CS-917 and MB07803; and the SMAD7 inhibitor mongersen, have been investigated in clinical trials. Interestingly, miR-26 better reduced intima-media thickness (IMT) than PCSK9 or CT-1 knockout. Many PCSK9 inhibitors, including alirocumab, evolocumab, inclisiran, AZD8233, Civi-007, MK-0616, and LIB003, have been investigated in clinical trials. Recombinant CT-1 was also investigated in clinical trials. Therefore, miR-26 is a promising target for agent development. miR-26 promotes foam cell formation by reducing ABCA1 and ARL4C expression. Multiple materials can be used to deliver miR-26, but it is unclear which material is most suitable for mass production and clinical applications. This review focuses on the potential use of miR-26 in treating atherosclerosis to support the development of agents targeting it.
Subject(s)
Atherosclerosis , MicroRNAs , Humans , ADP-Ribosylation Factors , Carotid Intima-Media Thickness , Diacylglycerol O-Acyltransferase , MicroRNAs/genetics , Proprotein Convertase 9 , Smad7 Protein , Atherosclerosis/geneticsABSTRACT
Gastric cancer (GC) is a highly aggressive malignancy with limited treatment options for advanced-stage patients. Recent studies have highlighted the role of circular RNA (circRNA) as a novel regulator of cancer progression in various malignancies. However, the underlying mechanisms by which circRNA contributes to the development and progression of GC remain poorly understood. In this study, we utilized microarrays and real-time quantitative polymerase chain reaction (qRT-PCR) to identify and validate a downregulated circRNA, hsa_circ_0003251 (referred to as circWNK1), in paired GC and normal tissues. Through a series of in vitro and in vivo gain-of-function and loss-of-function assays, we demonstrated that circWNK1 exerts inhibitory effects on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of GC cells. Additionally, we discovered that circWNK1 acts as a competitive endogenous RNA (ceRNA) for SMAD7 by sequestering miR-21-3p. Our findings were supported by comprehensive biological information analysis, as well as RNA pull-down, luciferase reporter gene, and western blot assays. Notably, the downregulation of circWNK1 in GC cells resulted in reduced SMAD7 expression, subsequently activating the TGF-ß signaling pathway. Collectively, our study reveals that circWNK1 functions as a tumor suppressor in GC by regulating the miR-21-3p/SMAD7-mediated TGF-ß signaling pathway. Furthermore, circWNK1 holds promise as a potential biomarker for the diagnosis and treatment of GC.
Subject(s)
MicroRNAs , Stomach Neoplasms , Humans , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/genetics , RNA, Circular/metabolism , Smad7 Protein/genetics , Smad7 Protein/metabolism , Stomach Neoplasms/pathology , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolismABSTRACT
Pulmonary fibrosis is a challenging lung disease characterized by a bleak prognosis. A pivotal element in the progression of this disease is the dysregulated recruitment of macrophages. Nicotinamide phosphoribose transferase (NAMPT), secreted by alveolar epithelial cells and inflammatory cells, has been previously identified to influence macrophage inflammation in acute lung injury through the nicotinamide adenine dinucleotide (NAD) rescue synthesis pathway. Nonetheless, the exact role of NAMPT in the regulation of lung fibrosis is yet to be elucidated. In our research, we employed bleomycin (BLM) to induce pulmonary fibrosis in Namptflox/flox;Cx3cr1CreER mice, using Namptflox/flox mice as controls. Our findings revealed an augmented expression of NAMPT concurrent with a marked increase in the secretion of NAD and inflammatory cytokines such as IL-6, TNF-α, and TGF-ß1 post-BLM treatment. Furthermore, an upsurge in NAMPT-positive macrophages was observed in the lungs of BLM-treated Namptflox/flox mice. Notably, a conditional knockout of NAMPT (NAMPT cKO) in lung macrophages curtailed the BLM-induced inflammatory responses and significantly mitigated pulmonary fibrosis. This was associated with diminished phospho-Sirt1 (p-Sirt1) expression levels and a concomitant rise in mothers against decapentaplegic homolog 7 (Smad7) expression in BLM-treated mouse lungs and murine RAW 264.7 macrophage cells. Collectively, our data suggests that NAMPT exacerbates macrophage-driven inflammation and pulmonary fibrosis via the Sirt1-Smad7 pathway, positioning NAMPT as a promising therapeutic target for pulmonary fibrosis intervention.