RESUMO
Atrial fibrosis is an important factor in the initiation and maintenance of atrial fibrillation (AF); therefore, understanding the pathogenesis of atrial fibrosis may reveal promising therapeutic targets for AF. In this study, we successfully established a rapid atrial pacing canine model and found that the inducibility and duration of AF were significantly reduced by the overexpression of c-Ski, suggesting that this approach may have therapeutic effects. c-Ski was found to be down-regulated in the atrial tissues of the rapid atrial pacing canine model. We artificially up-regulated c-Ski expression with a c-Ski-overexpressing adenovirus. Haematoxylin and eosin, Masson's trichrome and picrosirius red staining showed that c-Ski overexpression alleviated atrial fibrosis. Furthermore, we found that the expression levels of collagen III and α-SMA were higher in the groups of dogs subjected to right-atrial pacing, and this increase was attenuated by c-Ski overexpression. In addition, c-Ski overexpression decreased the phosphorylation of smad2, smad3 and p38 MAPK (p38α and p38ß) as well as the expression of TGF-ß1 in atrial tissues, as shown by a comparison of the right-atrial pacing + c-Ski-overexpression group to the control group with right-atrial pacing only. These results suggest that c-Ski overexpression improves atrial remodelling in a rapid atrial pacing canine model by suppressing TGF-ß1-Smad signalling and p38 MAPK activation.
Assuntos
Remodelamento Atrial , Estimulação Cardíaca Artificial , Átrios do Coração/fisiopatologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Modelos Animais de Doenças , Cães , Fenômenos Eletrofisiológicos , Matriz Extracelular/metabolismo , Fibrose , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Although transforming growth factor beta (TGF-ß) is known to be involved in the pathogenesis and progression of many cancers, its role in renal cancer has not been fully investigated. In the present study, we examined the role of TGF-ß in clear cell renal carcinoma (ccRCC) progression in vitro and in vivo. First, expression levels of TGF-ß signaling pathway components were examined. Microarray and immunohistochemical analyses showed that the expression of c-Ski, a transcriptional corepressor of Smad-dependent TGF-ß and bone morphogenetic protein (BMP) signaling, was higher in ccRCC tissues than in normal renal tissues. Next, a functional analysis of c-Ski effects was carried out. Bioluminescence imaging of renal orthotopic tumor models demonstrated that overexpression of c-Ski in human ccRCC cells promoted in vivo tumor formation. Enhancement of tumor formation was also reproduced by the introduction of a dominant-negative mutant TGF-ß type II receptor into ccRCC cells. In contrast, introduction of the BMP signaling inhibitor Noggin failed to accelerate tumor formation, suggesting that the tumor-promoting effect of c-Ski depends on the inhibition of TGF-ß signaling rather than of BMP signaling. Finally, the molecular mechanism of the tumor-suppressive role of TGF-ß was assessed. Although TGF-ß signaling did not affect tumor angiogenesis, apoptosis of ccRCC cells was induced by TGF-ß. Taken together, these findings suggest that c-Ski suppresses TGF-ß signaling in ccRCC cells, which, in turn, attenuates the tumor-suppressive effect of TGF-ß.
Assuntos
Carcinoma de Células Renais/genética , Proteínas de Ligação a DNA/genética , Neoplasias Renais/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transplante HeterólogoRESUMO
Cardiac fibrosis is characterized by over-deposition of extracellular matrix (ECM) proteins and over-proliferation of cardiac fibroblast, and contributes to both systolic and diastolic dysfunction in many cardiac pathophysiologic conditions. Transforming growth factor ß 1 (TGFß1) is as an essential inducing factor of cardiac fibrosis. C-Ski protein has been identified as an inhibitory regulator of TGFß signaling. In the present study, we revealed the repressive effect of c-Ski on TGFß1-induced human cardiac fibroblast (HCFB) proliferation and ECM protein increase (Collagen I and α-SMA). Moreover, miR-155 and miR-17 could inhibit SKI mRNA expression by direct binding to the 3'UTR of SKI, so as to reduce c-Ski protein level. Either miR-155 inhibition or miR-17 inhibition could reverse TGFß1-induced HCFB proliferation and ECM protein increase. Taken together, we provided a potential therapy to treat cardiac fibrosis by inhibiting miR-155/miR-17 so as to restore the repressive effect of c-Ski on TGFß1 signaling. J. Cell. Biochem. 118: 1911-1920, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Regiões 3' não Traduzidas/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Proteínas da Matriz Extracelular , Fibroblastos/efeitos dos fármacos , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Cancer-associated inflammation develops resistance to the epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancers (NSCLCs) harboring oncogenic EGFR mutations. Stat3-mediated interleukin (IL)-6 signaling and Smad-mediated transforming growth factor-ß (TGF-ß) signaling pathways play crucial regulatory roles in cancer-associated inflammation. However, mechanisms how these pathways regulate sensitivity and resistance to EGFR-TKI in NSCLCs remain largely undetermined. Here we show that signal transducer and activator of transcription (Stat)3 represses Smad3 in synergy with the potent negative regulators of TGF-ß signaling, c-Ski and SnoN, whereby renders gefitinib-sensitive HCC827 cells resistant. We found that IL-6 signaling via phosphorylated Stat3 induced gefitinib resistance as repressing transcription of Smad3, whereas TGF-ß enhanced gefitinib sensitivity as activating transcription of Smad3 in HCC827 cells with gefitinib-sensitizing EGFR mutation. Promoter analyses showed that Stat3 synergized with c-Ski/SnoN to repress Smad2/3/4-induced transcription of the Smad3 gene. Smad3 was found to be an apoptosis inducer, which upregulated pro-apoptotic genes such as caspase-3 and downregulated anti-apoptotic genes such as Bcl-2. Our results suggest that derepression of Smad3 can be a therapeutic strategy to prevent gefitinib-resistance in NSCLCs with gefitinib-sensitizing EGFR mutation.
Assuntos
Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Quinazolinas/farmacologia , Fator de Transcrição STAT3/fisiologia , Proteína Smad3/antagonistas & inibidores , Adenocarcinoma/patologia , Apoptose/fisiologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Gefitinibe , Humanos , Interleucina-6/metabolismo , Neoplasias Pulmonares/patologia , Mutação , Transdução de SinaisRESUMO
Smad proteins transduce signals downstream of transforming growth factor-ß (TGF-ß) and are one of the factors that regulate the expression of genes related to diseases affecting the skin. In the present study, we identified MAB21L4, also known as male abnormal 21 like 4 or C2orf54, as the most up-regulated targets of TGF-ß and Smad3 in differentiated human progenitor epidermal keratinocytes using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). We found that TGF-ß induced expression of the barrier protein involucrin (encoded by the IVL gene). Transcriptional activity of the IVL promoter induced by TGF-ß was inhibited by MAB21L4 siRNAs. Further analysis revealed that MAB21L4 siRNAs also down-regulated the expression of several target genes of TGF-ß. MAB21L4 protein was located mainly in the cytosol, where it was physically bound to Smad3 and a transcriptional corepressor c-Ski. siRNAs for MAB21L4 did not inhibit the binding of Smad3 to their target genomic regions but down-regulated the acetylation of histone H3 lys 27 (H3K27ac), an active histone mark, near the Smad3 binding regions. These findings suggest that TGF-ß-induced MAB21L4 up-regulates the gene expression induced by TGF-ß, possibly through the inhibition of c-Ski via physical interaction in the cytosol.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta , Humanos , Queratinócitos/metabolismo , Masculino , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Cardiovascular disease (CVD) is a leading non-communicable disease with a high fatality rate worldwide. Hypertension, a common cardiovascular condition, is a significant risk factor for the development of heart failure because the activation of the renin-angiotensin system (RAS) is considered to be the major promoting reason behind myocardial fibrosis (MF). In this study, Angiotensin II (Ang II) stimulation-induced endothelial to mesenchymal transition (End-MT) in HCAECs, including the decrease of CD31 level, the increase of α-SMA, collagen I, slug, snail, and TGF-ß1 levels, and the promotion of Smad2/3 phosphorylation. Meanwhile, the c-Ski level was reduced in Ang II-stimulated HCAECs. In HCAECs, Ang II-induced changes could be partially attenuated by c-Ski overexpression. miR-214-3p directly targeted c-Ski and inhibited c-Ski expression. Moreover, miR-214-3p inhibition reduced Ang II-caused End-MT in HCAECs. miR-214-3p overexpression further enhanced Ang II-induced End-MT, while c-Ski overexpression could markedly reverse the effects of miR-214-3p overexpression. In the Ang II-induced mouse cardiac hypertrophic model, Ang II-caused increase of cellular cross-sectional area and cardiac fibrosis were partially ameliorated by LV-c-Ski; when mice were co-treated with LV-c-Ski and agomir-214-3p, the beneficial effects of LV-c-Ski were reversed. In conclusion, the miR-214-3p/c-Ski axis modulated Ang II-induced End-MT in HCAECs and cardiac hypertrophy and fibrosis in the mice model.
Assuntos
Proteínas de Ligação a DNA , Células Endoteliais , MicroRNAs , Proteínas Proto-Oncogênicas , Animais , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Fibrose , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Transdução de SinaisRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Apigenin is a natural flavonoid compound present in chamomile (Matricaia chamomilla L.) from the Asteraceae family, which is used in the treatment of cardiovascular diseases by traditional healers, but its effects on differentiation and extracellular matrix (ECM) production of cardiac fibroblasts (CFs) induced by transforming growth factor beta 1 (TGF-ß1) are poorly understood. AIM OF THE STUDY: This study aimed to examine these effects and potential molecular mechanisms and to provide a new application of apigenin in the prevention and treatment of cardiac fibrosis. MATERIALS AND METHODS: The TGF-ß1-stimulated CFs or the combination of TGF-ß1-stimulated and microRNA-155-5p (miR-155-5p) inhibitor- or mimic-transfected CFs were treated with or without apigenin. The expression levels of intracellular related mRNA and proteins were detected by real-time polymerase chain reaction and Western blot methods, respectively. The luciferase reporter gene containing cellular Sloan-Kettering Institute (c-Ski) wild or mutant type 3'-UTR was used and the luciferase activity was examined to verify the direct link of miR-155-5p and c-Ski. RESULTS: After treatment of TGF-ß1-stimulated CFs with 6-24⯵M apigenin, the expression of c-Ski was increased, while levels of miR-155-5p, α-smooth muscle actin, collagen â /â ¢, Smad2/3, and p-Smad2/3 were decreased. After transfection of CFs with the miR-155-5p inhibitor or mimic, the similar or inverse results were respectively observed as well. The combination of TGF-ß1 and miR-155-5p inhibitor or mimic might cause an antagonistical or synergistic effect, respectively, and apigenin addition could enhance the effects of the inhibitor and antagonize the effects of the mimic. Luciferase reporter gene assay demonstrated that c-Ski was a direct target of miR-155-5p. CONCLUSION: These findings suggested that apigenin could inhibit the differentiation and ECM production in TGF-ß1-stimulated CFs, and its mechanisms might partly be attributable to the reduction of miR-155-5p expression and subsequent increment of c-Ski expression, which might result in the inhibition of Smad2/3 and p-Smad2/3 expressions.
Assuntos
Apigenina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Animais , Apigenina/isolamento & purificação , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Matricaria/química , Camundongos , MicroRNAs/genética , Miocárdio/citologia , Proteínas Proto-Oncogênicas/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The dysfunction of vascular smooth muscle cells (VSMCs) is critical for atherosclerosis (AS) progression. Autophagy is indispensable during phenotypic switching and proliferation of VSMCs, contribute to AS development. Cellular Sloan-Kettering Institute (c-Ski), the repressor of TGF-ß signaling, is involved in diverse physiological and pathological processes. We previously defined c-Ski also as an endogenous protective molecule against AS via inhibiting abnormal proliferation and autophagy of VSMCs. However, the endogenous level of c-Ski in VSMCs is markedly decreased during the progression of AS, so that the protective effect is drastically weakened. Elucidating the molecular mechanisms is key to the understanding of AS development and treatment. We determined that oxidized low-density lipoprotein (ox-LDL) and platelet-derived growth factor (PDGF) directly induced the degradation of c-Ski protein, closely associated with reducing its phosphorylation. Serine383 (S383) was identified as the crucial phosphorylation site for stabilizing protein expression and nuclear location of c-Ski, which was responsible for its transcriptional suppression of autophagy-related genes. Decreased S383 phosphorylation facilitated nuclear export and degradation of c-Ski, thereby lessened its inhibitory effect on induction of autophagy genes. These findings provide a novel view of c-Ski modification and function modulation under some vascular injury factors, which point to a new potential therapeutic strategy by targeting c-Ski.
Assuntos
Músculo Liso Vascular , Miócitos de Músculo Liso , Proliferação de Células , Células Cultivadas , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologiaRESUMO
Background: TGF-ß1 promotes cell proliferation in only some tumors and exerts bidirectional regulatory effects on the proliferation of fibroblasts. This study intends to explore whether the mechanism is related to increased expression of Ski. Methods: Cell proliferation of the fibrosarcoma cell line L929 was assessed with an ELISA BrdU kit. The mRNA and protein expression levels of the corresponding factors were measured by RT-qPCR, immunohistochemistry or Western blotting in vitro and in vivo. Additionally, c-Ski was knocked down using RNAi. The expression of Ski in human dermatofibrosarcoma protuberans (DFSP) specimens was measured by immunohistochemistry. Results: TGF-ß1 promoted the continued proliferation of L929 cells in a dose-dependent manner, with increased c-Ski expression levels. Conversely, inhibition of c-Ski significantly abrogated this unidirectional effect, significantly inhibited the decrease in p21 protein levels and did not affect the increase in p-Smad2/3 levels upon TGF-ß1 treatment. Similarly, inhibition of c-Ski significantly abrogated the growth-promoting effect of TGF-ß1 on xenograft tumors. Furthermore, we found that high expression of Ski in DFSP was correlated with a low degree of tumor differentiation. Conclusions: Our data reveal that high c-Ski expression is a cause of TGF-ß1-promoted proliferation in fibrosarcoma tumor cells and show that inhibiting Ski expression might be effective for treating tumors with high Ski levels.
RESUMO
The present study aimed to investigate the effects of c-Ski on cell proliferation, invasion and migration of gastric cancer associated fibroblasts (CAFs). Expression of c-Ski in gastric cancer (GC) tissues was determined using immunohistochemistry. Both CAFs and non-cancerous gastric fibroblasts (NGFs) were isolated and cultured. c-Ski and Smad3 were over-expressed or knocked down using pcDNA3.0-c-Ski/Smad3 or siRNA, respectively. Cell viability, invasion and migration were measured and expression of c-Ski, α-SMA, and Smad3 in cells was determined using real time quantitative PCR (RT-qPCR) and Western blotting. Expression of c-Ski was significantly higher in both in GC tissues and cell lines, and was the highest in tissues of diffuse type. Both c-Ski and α-SMA were significantly over-expressed in CAFs compared with that in the NGFs. When c-Ski was over-expressed in NGFs, cell viability, cell invasion and migration were all enhanced and expression of Smad3 was downregulated. When c-Ski was inhibited, cell viability, cell invasion, and migration were all suppressed and expression of Smad3 was upregulated. Meanwhile, overexpression of Smad3 significantly reversed the effects of over-expressed c-Ski in NGFs, and knockdown of Smad3 dramatically reversed the effects of si-c-Ski in CAFs. Over-expressed c-Ski could enhance cell viability, promote cell invasion, and migration of GC CAFs, and the effects might be through regulation of Smad3 signaling. This study may give deeper insights for relationship between c-Ski and CAFs, as well as role of c-Ski in cancer development, and also provide some novel research targets for treatment of GC.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Movimento Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais , Proteína Smad3/metabolismo , Regulação para Cima/genéticaRESUMO
BACKGROUND: The shRNA lentiviral vector was constructed to silence c-Ski expression in cardiac mus- cle cells, with the aim of exploring the role of c-Ski in transforming growth factor b1 (TGF-b1)-induced epithelial-mesenchymal transitions (EMT) in H9C2 cells. METHODS: Real-time polymerase chain reaction (RT-PCR) and western blot were used to detect c-Ski ex- pression at protein and messenger ribonucleic acid (mRNA) levels in 5 different cell lines. Then, lentiviral vector was constructed to silence or overexpress c-Ski in H9C2 cells. MTT and/or soft agar assay and tran- swell assay were used to detect cell proliferation and migration, respectively. The expression levels of c-Ski under different concentrations of TGF-b1 stimulation were detected by RT-qPCR and immunocytochemi- cal analysis. In the presence or absence of TGF-b1 stimulation, the proteins' expression levels of a-SMA, FN and E-cadherin, which are closely correlated with the process of EMT, were measured by western blot after c-Ski silencing or overexpression. Meanwhile, the effect of c-Ski on Samd3 phosphorylation with TGF-b1 stimulation was investigated. RESULTS: There is a high expression of c-Ski at protein and mRNA levels in H9C2 cell line, which first demonstrated the presence of c-Ski expression in H9C2 cells. Overexpression of c-Ski significantly increased H9C2 cell proliferation. The ability of c-Ski gene silencing to suppress cell proliferation was gradually enhanced, and inhibition efficiency was the highest after 6 to 7 d of transfection. Moreover, H9C2 cells with c-Ski knockdown gained significantly aggressive invasive potential when compared with the control group. TGF-b1 stimulation could dose-independently reduce c-Ski expression in H9C2 cells and lead to obvious down-regulated expression of E-cadherin. Interestingly, c-Ski could restore E-cadherin expression while suppressing a-SMA and/or FN expression stimulated by TGF-b1. How- ever, shRNA-induced c-Ski knockdown aggravated only the TGF-b1-induced EMT. Moreover, c-Ski- -shRNA also promoted the phosphorylation of Samd3 induced by TGF-b1. CONCLUSIONS: c-Ski expression in cardiac muscle cells could be down-regulated by TGF-b1. Silencing of c-Ski gene was accompanied by down-regulation of E-cadherin, up-regulation of a-SMA and/or FN and Smad3 phosphorylation induced by TGF-b1, promoting EMT process. Therefore, c-Ski may be closely associated with TGF-b1-induced EMT and play an important role in cardiac fibrosis develop- ment and progression.
Assuntos
Cardiomiopatias/genética , Proteínas de Ligação a DNA/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação da Expressão Gênica , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA/genética , Fator de Crescimento Transformador beta1/metabolismo , Western Blotting , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Linhagem Celular , Proliferação de Células , Proteínas de Ligação a DNA/biossíntese , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Inativação Gênica , Humanos , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas/biossíntese , Transdução de SinaisRESUMO
Increasing evidence has suggested that bidirectional regulation of cell proliferation is one important effect of TGF-ß1 in wound healing. Increased c-Ski expression plays a role in promoting fibroblast proliferation at low TGF-ß1 concentrations, but the mechanism by which low TGF-ß1 concentrations regulate c-Ski levels remains unclear. In this study, the proliferation of rat primary fibroblasts was assessed with an ELISA BrdU kit. The mRNA and protein expression and phosphorylation levels of corresponding factors were measured by RT-qPCR, immunohistochemistry or Western blotting. We first found that low TGF-ß1 concentrations not only promoted c-ski mRNA and protein expression in rat primary fibroblasts but also increased the phosphorylation levels of Extracellular Signal-Regulated Kinases (ERK) and cAMP response element binding (CREB) protein. An ERK kinase (mitogen-activated protein kinase kinase, MEK) inhibitor significantly inhibited ERK1/2 phosphorylation levels, markedly reducing c-Ski expression and CREB phosphorylation levels and abrogating the growth-promoting effect of low TGF-ß1 concentrations. At the same time, Smad2/3 phosphorylation levels were not significantly changed. Taken together, these results suggest that the increased cell proliferation induced by low TGF-ß1 concentrations mediates c-Ski expression potentially through the ERK/CREB pathway rather than through the classic TGF-ß1/Smad pathway.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/citologia , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fosforilação/efeitos dos fármacos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Pele/citologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismoRESUMO
Cardiac fibrosis is associated with diverse heart diseases. In response to different pathological irritants, cardiac fibroblasts may be induced to proliferate and differentiate into cardiac myofibroblasts, thus contributing to cardiac fibrosis. TGF-ß signaling is implicated in the development of heart failure through the induction of cardiac fibrosis. C-Ski, an inhibitory regulator of TGF-ß signaling, has been reported to suppress TGF-ß1-induced human cardiac fibroblasts' proliferation and ECM protein increase; however, the underlying molecular mechanism needs further investigation. In the present study, we demonstrated that c-Ski could ameliorate isoproterenol (ISO)-induced rat myocardial fibrosis model and TGF-ß1-induced primary rat cardiac fibroblasts' proliferation, as well as extracellular matrix (ECM) deposition. The protein level of c-Ski was dramatically decreased in cardiac fibrosis and TGF-ß1-stimulated primary rat cardiac fibroblasts. In recent decades, a family of small non-coding RNA, namely miRNAs, has been reported to regulate gene expression by interacting with diverse mRNAs and inducing either translational suppression or mRNA degradation. Herein, we selected miR-34a and miR-93 as candidate miRNAs that might target to regulate c-Ski expression. After confirming that miR-34a/miR-93 targeted c-Ski to inhibit its expression, we also revealed that miR-34a/miR-93 affected TGF-ß1-induced fibroblasts' proliferation and ECM deposition through c-Ski. Taken together, we demonstrated a miR-34a/miR-93-c-Ski axis which modulates TGF-ß1- and ISO-induced cardiac fibrosis in vitro and in vivo; targeting the inhibitory factors of c-Ski to rescue its expression may be a promising strategy for the treatment of cardiac fibrosis.
Assuntos
MicroRNAs/fisiologia , Miocárdio/patologia , Miofibroblastos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proliferação de Células , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibrose , Células HEK293 , Humanos , Masculino , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Human coronary artery endothelial cells (HCAECs) have the potential to undergo fibrogenic endothelial-mesenchymal transition (EndMT), which results in matrix-producing fibroblasts and thereby contributes to the pathogenesis of cardiac fibrosis. Recently, the profibrotic cytokine transforming growth factor-ß (TGF-ß) is shown to be the crucial pathogenic driver which has been verified to induce EndMT. C-Ski is an important regulator of TGF-ß signaling. However, the detailed role of c-Ski and the molecular mechanisms by which c-Ski affects TGF-ß-induced EndMT in HCAECs are not largely elucidated. In the present study, we treated HCAECs with TGF-ß of different concentrations to induce EndMT. We found that overexpression of c-Ski in HCAECs either blocked EndMT via hindering Vimentin, Snail, Slug, and Twist expression while enhancing CD31 expression, with or without TGF-ß treatment. In contrast, suppression of c-Ski further enhanced EndMT. Currently, miRNA expression disorder has been frequently reported associating with cardiac fibrosis. By using online tools, we regarded miR-155 as a candidate miRNA that could target c-Ski, which was verified using luciferase assays. C-Ski expression was negatively regulated by miR-155. TGF-ß-induced EndMT was inhibited by miR-155 silence; the effect of TGF-ß on Vimentin, CD31, Snail, Slug, and Twist could be partially restored by miR-155. Altogether, these findings will shed light on the role and mechanism by which miR-155 regulates TGF-ß-induced HCAECs EndMT via c-Ski to affect cardiac fibrosis, and miR-155/c-Ski may represent novel biomarkers and therapeutic targets in the treatment of cardiac fibrosis.
Assuntos
Vasos Coronários/citologia , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Biomarcadores , Linhagem Celular , Proteínas de Ligação a DNA/genética , Fibrose , Humanos , MicroRNAs/genética , Miocárdio/patologia , Proteínas Proto-Oncogênicas/genéticaRESUMO
Previously, we reported that the decrease of endogenous c-Ski expression is implicated in the progression of vascular smooth muscle cell (VSMC) proliferation after arterial injury. However, the molecular mechanism of the down-regulation of c-Ski is not clear. In this study, a potential miR-21 recognition element was identified in the 3'-untranslated region (UTR) of rat c-Ski mRNA. A reporter assay revealed that miR-21 could recognize the miR-21 recognition element of c-Ski mRNA. In A10 rat aortic smooth muscle cells, overexpression of miR-21 significantly inhibited the expression of c-Ski protein and promoted cell proliferation, which could be blocked by inhibition of miR-21 or overexpression of c-Ski. Further investigation demonstrated that the effect of miR-21 on VSMC proliferation resulted from negative regulation of c-Ski to suppress p38-p21/p27 signaling, the downstream pathway of c-Ski in VSMCs. These results indicate that c-Ski is a target gene of miR-21. miR-21 specifically binds to the 3'-untranslated region of c-Ski and negatively regulates c-Ski expression to diminish the protective effects of c-Ski and stimulate VSMC proliferation in the progression of arterial injury.
Assuntos
MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Regiões 3' não Traduzidas , Animais , Aorta/citologia , Aorta/lesões , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Masculino , MicroRNAs/antagonistas & inibidores , Músculo Liso Vascular/citologia , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Aberrant expression of c-Ski oncoprotein in some tumor cells has been shown to be associated with cancer development. However, the role of c-Ski in cancer-associated fibroblasts (CAFs) of tumor microenvironment has not been characterized. In the current study, we found that c-Ski is highly expressed in CAFs derived from breast carcinoma microenvironment and this CAF-associated c-Ski expression is associated with invasion and metastasis of human breast tumors. We showed that c-Ski overexpression in immortalized breast normal fibroblasts (NFs) induces conversion to breast CAFs by repressing p53 and thereby upregulating SDF-1 in NFs. SDF-1 treatment or p53 knockdown in NFs had similar effects on the activation of NFs as c-Ski overexpression. The c-Ski-activated CAFs show increased proliferation, migration, invasion and contraction compared with NFs. Furthermore, c-Ski-activated CAFs facilitated the migration and invasion of MDA-MB-231 breast cancer cells. Our data suggest that c-Ski is an important regulator in the activation of CAFs and may serve as a potential therapeutic target to block breast cancer progression.