RESUMO
In lactating mothers, the high calcium (Ca2+) demand for milk production triggers significant bone loss1. Although oestrogen normally counteracts excessive bone resorption by promoting bone formation, this sex steroid drops precipitously during this postpartum period. Here we report that brain-derived cellular communication network factor 3 (CCN3) secreted from KISS1 neurons of the arcuate nucleus (ARCKISS1) fills this void and functions as a potent osteoanabolic factor to build bone in lactating females. We began by showing that our previously reported female-specific, dense bone phenotype2 originates from a humoral factor that promotes bone mass and acts on skeletal stem cells to increase their frequency and osteochondrogenic potential. This circulatory factor was then identified as CCN3, a brain-derived hormone from ARCKISS1 neurons that is able to stimulate mouse and human skeletal stem cell activity, increase bone remodelling and accelerate fracture repair in young and old mice of both sexes. The role of CCN3 in normal female physiology was revealed after detecting a burst of CCN3 expression in ARCKISS1 neurons coincident with lactation. After reducing CCN3 in ARCKISS1 neurons, lactating mothers lost bone and failed to sustain their progeny when challenged with a low-calcium diet. Our findings establish CCN3 as a potentially new therapeutic osteoanabolic hormone for both sexes and define a new maternal brain hormone for ensuring species survival in mammals.
Assuntos
Densidade Óssea , Osso e Ossos , Encéfalo , Hormônios , Mães , Proteína Sobre-Expressa em Nefroblastoma , Osteogênese , Adolescente , Animais , Feminino , Humanos , Masculino , Camundongos , Envelhecimento , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/metabolismo , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Remodelação Óssea , Reabsorção Óssea/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Cálcio/administração & dosagem , Cálcio/metabolismo , Lactação/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Células-Tronco/metabolismo , Células-Tronco/citologia , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Hormônios/metabolismoRESUMO
INTRODUCTION: Childhood obesity is a global health problem that is associated with various metabolic complications, such as insulin resistance, type 2 diabetes, dyslipidemia, and cardiovascular diseases. The mechanisms underlying the development of insulin resistance in childhood obesity are not fully understood. Nephroblastoma overexpressed gene (NOV), also known as CCN3, is a member of the CCN family of matricellular proteins that modulate cell proliferation, differentiation, adhesion, migration, and survival. Previous studies have shown that NOV/CCN3 is involved in glucose metabolism and insulin signaling in various tissues and cell types. However, the role of NOV/CCN3 in childhood obesity and insulin resistance remains unclear. METHODS: In this study, we aimed to investigate the association between plasma NOV/CCN3 levels and insulin resistance in 58 obese and 43 non-obese children aged 6-12 years. We measured plasma NOV/CCN3 levels by enzyme-linked immunosorbent assay and assessed insulin resistance by homeostasis model assessment of insulin resistance (HOMA-IR). We also collected clinical and biochemical data, such as body mass index (BMI), waist circumference (WC), blood pressure (BP), fasting glucose (FG), fasting insulin (FI), lipid profile, and inflammatory markers. RESULTS: We found that plasma NOV/CCN3 levels were significantly higher in obese children than in non-obese children (p < 0.001) and positively correlated with BMI (r = 0.42, p < 0.001), WC (r = 0.38, p < 0.001), BP (r = 0.35, p < 0.001), FG (r = 0.31, p < 0.001), FI (r = 0.45, p < 0.001), HOMA-IR (r = 0.48, p < 0.001), triglycerides (r = 0.28, p < 0.001), low-density lipoprotein cholesterol (r = 0.26, p < 0.001), and C-reactive protein (CRP) (r = 0.32, p < 0.001). Multiple linear regression analysis revealed that plasma NOV/CCN3 levels were independently associated with HOMA-IR after adjusting for age, sex, BMI, WC, BP, FG, FI, lipid profile, and CRP (ß = 0.36, p < 0.001). CONCLUSION: These results suggest that plasma NOV/CCN3 levels are elevated in childhood obesity and are associated with insulin resistance, indicating that NOV/CCN3 may play a role in the pathogenesis of metabolic disorders in obese children.
Assuntos
Índice de Massa Corporal , Resistência à Insulina , Proteína Sobre-Expressa em Nefroblastoma , Obesidade Infantil , Humanos , Proteína Sobre-Expressa em Nefroblastoma/sangue , Masculino , Feminino , Obesidade Infantil/sangue , Criança , Glicemia/análise , Circunferência da Cintura , Insulina/sangue , Biomarcadores/sangue , Estudos Transversais , Pressão SanguíneaRESUMO
Neural stem/progenitor cells (NSPCs) hold immense promise in clinical applications, yet the harsh conditions resulting from central nervous system (CNS) injuries, particularly oxidative stress, lead to the demise of both native and transplanted NSPCs. Cellular communication network factor 3 (CCN3) exhibits a protective effect against oxidative stress in various cell types. This study investigates the impact of CCN3 on NSPCs apoptosis induced by oxidative stress. To establish models of primary cultured mouse NSPCs under oxidative stress, we exposed them to 50 µM H2O2 for 4 h. Remarkably, pre-exposing CCN3 exacerbated the H2O2-induced decline in cell viability in a concentration-dependent manner. However, employing gene-targeted siRNA to inhibit CCN3 protected NSPCs against H2O2-induced cell death. Conversely, CCN3 replenishment reversed this protective effect, as evidenced by TUNEL staining, the ratio of Cleaved-caspase-3 to Pro-caspase-3, and Bcl-2/Bax. Further investigations revealed that CCN3 pretreatment increased the phosphorylation level of p38 MAPK, while silencing CCN3 diminished p38 MAPK activation. Ultimately, the impact of changes in CCN3 protein expression on H2O2-induced apoptosis was nullified using anisomycin (a p38 activator) and SB 203580 (a p38 inhibitor). Our findings suggest that CCN3 inhibition prevents H2O2-induced cell death in cultured mouse NSPCs via the p38 pathway. These discoveries may contribute to the development of strategies aimed at enhancing the survival of both endogenous and transplanted NSPCs following CNS oxidative stress insults.
Assuntos
Peróxido de Hidrogênio , Proteínas Quinases p38 Ativadas por Mitógeno , Camundongos , Animais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/farmacologia , Estresse Oxidativo , Apoptose , Células-Tronco/metabolismoRESUMO
Neural stem cells (NSCs) persist in the subgranular zone (SGZ) throughout the lifespan and hold immense potential for the repair and regeneration of the central nervous system, including hippocampal-related diseases. Several studies have demonstrated that cellular communication network protein 3 (CCN3) regulates multiple types of stem cells. However, the role of CCN3 in NSCs remains unknown. In this study, we identified CCN3 expression in mouse hippocampal NSCs and observed that supplementing CCN3 improved cell viability in a concentration-dependent manner. Additionally, in vivo results showed that the injection of CCN3 in the dentate gyrus (DG) increased Ki-67- and SOX2-positive cells while decreasing neuron-specific class III beta-tubulin (Tuj1) and doublecortin (DCX)-positive cells. Consistently with the in vivo results, supplementing CCN3 in the medium increased the number of BrdU and Ki-67 cells and the proliferation index but decreased the number of Tuj1 and DCX cells. Conversely, both the in vivo and in vitro knockdown of the Ccn3 gene in NSCs had opposite effects. Further investigations revealed that CCN3 promoted cleaved Notch1 (NICD) expression, leading to the suppression of PTEN expression and eventual promotion of AKT activation. In contrast, Ccn3 knockdown inhibited the activation of the Notch/PTEN/AKT pathway. Finally, the effects of changes in CCN3 protein expression on NSC proliferation and differentiation were eliminated by FLI-06 (a Notch inhibitor) and VO-OH (a PTEN inhibitor). Our findings imply that while promoting proliferation, CCN3 inhibits the neuronal differentiation of mouse hippocampal NSCs and that the Notch/PTEN/AKT pathway may be a potential intracellular target of CCN3. Our findings may help develop strategies to enhance the intrinsic potential for brain regeneration after injuries, particularly stem cell treatment for hippocampal-related diseases.
Assuntos
Proteína Sobre-Expressa em Nefroblastoma , Células-Tronco Neurais , Proteínas Proto-Oncogênicas c-akt , Animais , Camundongos , Diferenciação Celular , Proliferação de Células , Hipocampo/metabolismo , Antígeno Ki-67/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Aberrant vascular remodeling contributes to the progression of many aging-associated diseases, including idiopathic pulmonary fibrosis (IPF), where heterogeneous capillary density, endothelial transcriptional alterations, and increased vascular permeability correlate with poor disease outcomes. Thus, identifying disease-driving mechanisms in the pulmonary vasculature may be a promising strategy to limit IPF progression. Here, we identified Ccn3 as an endothelial-derived factor that is upregulated in resolving but not in persistent lung fibrosis in mice, and whose function is critical for vascular homeostasis and repair. Loss and gain of function experiments were carried out to test the role of CCN3 in lung microvascular endothelial function in vitro through RNAi and the addition of recombinant human CCN3 protein, respectively. Endothelial migration, permeability, proliferation, and in vitro angiogenesis were tested in cultured human lung microvascular endothelial cells (ECs). Loss of CCN3 in lung ECs resulted in transcriptional alterations along with impaired wound-healing responses, in vitro angiogenesis, barrier integrity as well as an increased profibrotic activity through paracrine signals, whereas the addition of recombinant CCN3 augmented endothelial function. Altogether, our results demonstrate that the matricellular protein CCN3 plays an important role in lung endothelial function and could serve as a promising therapeutic target to facilitate vascular repair and promote lung fibrosis resolution.
Assuntos
Fibrose Pulmonar , Camundongos , Humanos , Animais , Células Endoteliais/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Células Cultivadas , Pulmão/metabolismoRESUMO
Cellular communication network factor (CCN) 2 and 3 are the members of the CCN family that conduct the harmonized development of a variety of tissues and organs under interaction with multiple biomolecules in the microenvironment. Despite their striking structural similarities, these two members show contrastive molecular functions as well as temporospatial emergence in living tissues. Typically, CCN2 promotes cell growth, whereas CCN3 restrains it. Where CCN2 is produced, CCN3 disappears. Nevertheless, these two proteins collaborate together to execute their mission in a yin-yang fashion. The apparent functional counteractions of CCN2 and CCN3 can be ascribed to their direct molecular interaction and interference over the cofactors that are shared by the two. Recent studies have revealed the mutual negative regulation systems between CCN2 and CCN3. Moreover, the simultaneous and bidirectional regulatory system of CCN2 and CCN3 is also being clarified. It is of particular note that these regulations were found to be closely associated with glycolysis, a fundamental procedure of energy metabolism. Here, the molecular interplay and metabolic gene regulation that enable the yin-yang collaboration of CCN2 and CCN3 typically found in cartilage development/regeneration and fibrosis are described.
Assuntos
Proteína Sobre-Expressa em Nefroblastoma , Yin-Yang , Proliferação de Células , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibrose , Regulação da Expressão Gênica , Humanos , Proteína Sobre-Expressa em Nefroblastoma/genética , Proteína Sobre-Expressa em Nefroblastoma/metabolismoRESUMO
Intrahepatic cholangiocarcinoma (ICC) is a common type of human cancer with a poor prognosis, and investigating the potential molecular mechanisms that can contribute to gene diagnosis and therapy. Herein, based on the recently concerned vertebrate-specific Cyr61/CTGF/NOV (CCN) gene family because of its important roles in diverse diseases, we obtained NOV/CCN3 to query for its potential roles in tumorigenesis via bioinformatics analysis. Experimental validations confirmed that both NOV mRNA and protein are up-regulated in two ICC cell lines, suggesting that it may promote cell migration and invasion by promoting EMT. To elucidate the detailed regulatory mechanism, miR-92a-3p is screened and identified as a negative regulatory small RNA targeting NOV, and further experimental validation demonstrates that miR-92a-3p contributes to NOV-mediated migration and invasion of ICC via the Notch signaling pathway. Our study reveals that NOV may be a potential target for diagnosing and treating ICC, which will provide experimental data and molecular theoretical foundation for cancer treatment, particularly for future precision medicine.
Assuntos
Neoplasias dos Ductos Biliares/patologia , Movimento Celular/genética , Colangiocarcinoma/patologia , MicroRNAs/fisiologia , Proteína Sobre-Expressa em Nefroblastoma/fisiologia , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos/efeitos dos fármacos , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Colangiocarcinoma/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , Invasividade Neoplásica , Proteína Sobre-Expressa em Nefroblastoma/genética , RNA Interferente Pequeno/farmacologiaRESUMO
The CCN proteins are a family of extracellular matrix- (ECM-) associated proteins which currently consist of six secreted proteins (CCN1-6). CCN3 protein, also known as nephroblastoma overexpressed protein (NOV), is a member of the CCN family with multiple biological functions, implicated in major cellular processes such as cell growth, migration, and differentiation. Recently, CCN3 has emerged as a critical regulator in a variety of diseases, including immune-related diseases, including rheumatology arthritis, osteoarthritis, and systemic sclerosis. In this review, we will briefly introduce the structure and function of the CCN3 protein and summarize the roles of CCN3 in immune-related diseases, which is essential to understand the functions of the CCN3 in immune-related diseases.
Assuntos
Sistema Imunitário/fisiopatologia , Proteína Sobre-Expressa em Nefroblastoma/fisiologia , Animais , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Diferenciação Celular , Movimento Celular , Proliferação de Células , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Fibrose , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Humanos , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Osteoartrite/imunologia , Osteoartrite/metabolismo , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/metabolismoRESUMO
BACKGROUND: Resveratrol improves insulin-resistance (IR) of gestational diabetes mellitus (GDM) mice. Low-expressed miR-23a-3p in diabetes patients regulates IR of adipocytes. Hence, we speculated the effect of Res on GDM mice was realized through regulating miR-23a-3p. METHODS: The GDM model was established in mice by high-fat diet, treated with miR-23a-3p antagomiR, and further performed with glucose and insulin tolerance tests. The bodyweight, serum glucose and serum insulin, and the expressions of miR-23a-3p and nephroblastoma overexpressed (NOV) in mouse adipose tissues were detected. MiR-23a-3p target was identified by Starbase and dual-luciferase reporter. Then, an IR adipocyte model was established by dexamethasone-inducing and further treated with Resveratrol or transfected with miR-23a-3p inhibitor or siNOV. The cell glucose intake was detected by radioimmunoassay. The expressions of miR-23a-3p, NOV, Adiponectin, Leptin, p-PI3K, PI3K, p-Akt, and Akt in the adipocytes were determined by qPCR or Western blot. RESULTS: Resveratrol decreased bodyweight, glucose level, insulin level, and the expressions of miR-23a-3p and NOV in the GDM mice, which was reversed by miR-23a-3p antagomiR. MiR-23a-3p targeted NOV. Resveratrol increased the glucose intake and the expressions of miR-23a-3p, Adiponectin, Leptin, p-PI3K, and p-Akt, decreased NOV expression in the IR adipocytes. The effect of the miR-23a-3p inhibitor on adipocytes with IR was opposite to Resveratrol, and the effects siNOV was the same as Resveratrol, except for its effect on miR-23a-3p expression. Effect of Res on the adipocytes with IR was counteracted by miR-23a-3p inhibitor whose effect was reversed by siNOV. CONCLUSION: Resveratrol ameliorated glucose uptake and lipid metabolism of the GDM mice and adipocytes with IR by regulating miR-23a-3p/NOV axis.
Assuntos
Adipócitos/efeitos dos fármacos , Diabetes Gestacional/metabolismo , Glucose/metabolismo , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/efeitos dos fármacos , MicroRNAs/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Resveratrol/farmacologia , Adipócitos/metabolismo , Animais , Diabetes Mellitus/metabolismo , Diabetes Mellitus Experimental/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Insulina/metabolismo , Camundongos , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tumor de Wilms/tratamento farmacológico , Tumor de Wilms/metabolismoRESUMO
BACKGROUND: Osteosarcoma is the most prevalent type of primary bone sarcoma and is the major cause of deaths associated with cancer in children and adolescents. Despite novel and innovative therapies, early diagnosis of the osteosarcoma is still critically needed. Our study aimed to analyse the CCN3 proteins as a diagnostic marker and correlate their expression level with the severity of primary osteosarcoma patients. METHODS: In this prospective case-control study, after ethical clearance and informed consent, a total of 35 cases with primary osteosarcoma and ten otherwise healthy controls were enroled according to our strict inclusion-exclusion criteria. Tissue samples were collected during biopsy procedures in suspected cases and in controls during bone grafting procedures. The CCN3 expression level was measured by the western blotting assay. The clinic-radiological examinations were done in cases and graded according to the AJCC classification. Comparisons of CCN3 expression were measured between cases and controls, followed by correlation of their expression level with severity/grade of osteosarcoma in cases. RESULTS: All the demographic parameters showed insignificant differences. The CCN3 protein expressions were significantly upregulated in tissue samples of osteosarcoma patients (cases) compared to controls. The mean difference (p<0.0001) in CCN3 protein expression between cases' and controls' bony tissues was significant but showed insignificant correlation with the different grades of osteosarcoma. CONCLUSIONS: The upregulated CCN3 protein expression in osteosarcoma tissue along with significant differential manifestation in accordance with different grades of osteosarcoma make CCN3 suitable for a potential diagnostic biomarker. However, the author recommends further extensive multi-centric collaborative studies to increase our study reliability and generalizability.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Osteossarcoma/diagnóstico , Adolescente , Adulto , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/cirurgia , Estudos de Casos e Controles , Criança , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Osteossarcoma/cirurgia , Índice de Gravidade de Doença , Tomografia Computadorizada por Raios X , Adulto JovemRESUMO
Cellular communication network factor (CCN) family members are multifunctional matricellular proteins that manipulate and integrate extracellular signals. In our previous studies investigating the role of CCN family members in cellular metabolism, we found three members that might be under the regulation of energy metabolism. In this study, we confirmed that CCN2 and CCN3 are the only members that are tightly regulated by glycolysis in human chondrocytic cells. Interestingly, CCN3 was induced under a variety of impaired glycolytic conditions. This CCN3 induction was also observed in two breast cancer cell lines with a distinct phenotype, suggesting a basic role of CCN3 in cellular metabolism. Reporter gene assays indicated a transcriptional regulation mediated by an enhancer in the proximal promoter region. As a result of analyses in silico, we specified regulatory factor binding to the X-box 1 (RFX1) as a candidate that mediated the transcriptional activation by impaired glycolysis. Indeed, the inhibition of glycolysis induced the expression of RFX1, and RFX1 silencing nullified the CCN3 induction by impaired glycolysis. Subsequent experiments with an anti-CCN3 antibody indicated that CCN3 supported the survival of chondrocytes under impaired glycolysis. Consistent with these findings in vitro, abundant CCN3 production by chondrocytes in the deep zones of developing epiphysial cartilage, which are located far away from the synovial fluid, was confirmed in vivo. Our present study uncovered that RFX1 is the mediator that enables CCN3 induction upon cellular starvation, which may eventually assist chondrocytes in retaining their viability, even when there is an energy supply shortage.
Assuntos
Condrócitos/metabolismo , Glicólise , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Fator Regulador X1/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Condrócitos/efeitos dos fármacos , Regulação da Expressão Gênica , Idade Gestacional , Glicólise/efeitos dos fármacos , Humanos , Articulações/embriologia , Articulações/metabolismo , Camundongos Endogâmicos BALB C , Proteína Sobre-Expressa em Nefroblastoma/genética , Fator Regulador X1/genética , Fluoreto de Sódio/farmacologiaRESUMO
We investigated the expression levels of nephroblastoma overexpressed [NOV or CCN3 (cellular communication network factor 3)] in the serum and placenta of pregnant women and of pregnant mice fed a high-fat diet (HFD), and its effect on placental glucose transporter 3 (GLUT3) expression, to examine its role in gestational diabetes mellitus (GDM). NOV/CCN3 expression was increased in the mouse serum during pregnancy. At gestational day 18, NOV/CCN3 protein expression was increased in the serum and placenta of the HFD mice compared with that of mice fed a normal diet. Compared with non-GDM patients, the patients with GDM had significantly increased serum NOV/CCN3 protein expression and placental NOV/CCN3 mRNA expression. Therefore, we hypothesized that NOV/CCN3 signaling may be involved in the pathogenesis of GDM. We administered NOV/CCN3 recombinant protein via intraperitoneal injections to pregnant mice fed HFD or normal diet. NOV/CCN3 overexpression led to glucose intolerance. Combined with the HFD, NOV/CCN3 exacerbated glucose intolerance and caused insulin resistance. NOV/CCN3 upregulates GLUT3 expression and affects the mammalian target of rapamycin (mTOR) pathway in the GDM environment in vivo and in vitro. In summary, our results demonstrate, for the first time, the molecular mechanism of NOV/CCN3 signaling in maternal metabolism to regulate glucose balance during pregnancy. NOV/CCN3 may be a potential target for detecting and treating GDM.NEW & NOTEWORTHY NOV/CCN3 regulates glucose homeostasis in mice during pregnancy. NOV/CCN3 upregulates GLUT3 expression and affects the mTOR pathway in the GDM environment in vivo and in vitro.
Assuntos
Dieta Hiperlipídica , Transportador de Glucose Tipo 3/genética , Proteína Sobre-Expressa em Nefroblastoma/genética , Serina-Treonina Quinases TOR/metabolismo , Animais , Células Cultivadas , Diabetes Gestacional/genética , Diabetes Gestacional/metabolismo , Gorduras na Dieta/farmacologia , Feminino , Glucose/metabolismo , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Humanos , Fenômenos Fisiológicos da Nutrição Materna/efeitos dos fármacos , Fenômenos Fisiológicos da Nutrição Materna/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Gravidez , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
OBJECTIVE: To investigate the role of NOV/CCN3 in regulating the proliferation of mesenchymal stem cells (MSCs) and its regulatory mechanism and assess the value of CCN3 as a proliferative factor in bone tissue engineering. METHODS: Mouse embryonic fibroblasts (MEFs) were used as the MSC model, in which CCN3 expression was up-regulated and downregulated by transfection with the recombinant adenovirus vectors Ad-CCN3 and Ad-siCCN3, respectively. Flow cytometry was used to analyze the changes in cell cycle and apoptosis of the transfected cells. Western blotting was used to detect the expression levels of the proliferation indicators (PCNA, cyclin E, and cyclin B1) and the apoptosis indicators (Bax and Bcl-2) to assess the effect of modulation of CCN3 expression on MEF proliferation and apoptosis. CCN3 protein secretion by the cells was detected using ELISA. RT-qPCR and Western blotting were employed to analyze the changes in the expressions of Notch1, ligand DLL1, the downstream key proteins or genes (Hey1, P300, H3K9) and MAPK pathway-related proteins ERK1+2 and p-ERK1+2. RESULTS: Flow cytometry showed that compared with the control cells, MEFs transfected with Ad-CCN3 exhibited significantly increased cell proliferation index (P < 0.01) and lowered cell apoptosis rate (P < 0.05) with obviously enhanced expressions of PCNA, cyclin E and Bcl-2 proteins (P < 0.05). The results of RT-qPCR and Western blotting demonstrated that CCN3 overexpression significantly promoted the expression of Notch1 in the Notch signaling pathway (P < 0.001), inhibited the expressions of DLL1, Hey1, P300, and H3K9 (P < 0.05), and increased the protein expressions of ERK1+2 and P-ERk1+2 in the MAPK pathway (P < 0.01). CONCLUSIONS: CCN3 over-expression promotes the proliferation and inhibits apoptosis of MEFs possibly by inhibiting the classical Notch signaling pathway and activating the MAPK pathway via binding to Notch1, suggesting the potential value of CCN3 as a proliferative factor of MSCs in bone tissue engineering.
Assuntos
Apoptose , Proteína Sobre-Expressa em Nefroblastoma , Animais , Ciclo Celular , Proliferação de Células , Fibroblastos , CamundongosRESUMO
Prostate cancer osteoblastic bone metastases are incurable and associated with chronic bone pain and a high mortality rate. Osteoclast-targeting drugs intended to prevent skeletal-related events associated with prostate cancer bone metastases do not prolong overall survival. Improved understanding of the bone-derived factors that contribute to prostate cancer osteoblastic bone metastases is required to design treatments that will improve morbidities and overall survival. Activated osteoblasts stimulate prostate cancer growth in bone. In this study, we report that prostate cancer conditioned medium (CM) promoted bone morphogenetic protein (BMP)-2, -4 and -7 production and the expression of osteogenic transcription factors Runx2 and osterix in osteoblasts. Treating the prostate cancer CM with antibody against CCN3 (nephroblastoma-overexpressed), a cysteine-rich protein that belongs to the CCN family, reduced all of these increases. Incubation of osteoblasts with CCN3 facilitated phosphorylation of GSK3ß and ß-catenin. GSK3ß and ß-catenin inhibitors or siRNAs all abolished CCN3-induced promotion of BMPs, Runx2 and osterix expression in osteoblasts. Our results indicate that prostate cancer-secreted CCN3 enhances BMP, Runx2 and osterix expression in osteoblasts via the GSK3ß and ß-catenin signaling pathways. This understanding of the role played by CCN3 in osteoblastic prostate bone metastasis may lead to more efficient targeted therapies.
Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Neoplasias da Próstata/metabolismo , beta Catenina/metabolismo , Animais , Neoplasias Ósseas/patologia , Diferenciação Celular , Humanos , Masculino , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Fosforilação , Transdução de SinaisRESUMO
Among cancer cells, there are specific cell populations of whose activities are comparable to those of stem cells in normal tissues, and for whom the levels of cell dedifferentiation are reported to correlate with poor prognosis. Information concerning the mechanisms that modulate the stemness like traits of cancer cells is limited. Therefore, we examined five gastric cancer cell lines and isolated gastric oncospheres from three gastric cancer cell lines. The gastric cancer cells that expanded in the spheres expressed relatively elevated proportion of CD44, which is a marker of gastric cancer stem cells (CSCs), and displayed many properties of CSCs, for example: chemoresistance, tumorigenicity and epithelial-mesenchymal transition (EMT) acquisition. SNAIL, which is a key factor in EMT, was highly expressed in the gastric spheres. Microarray analysis in gastric cancer cell line HGC27 showed that CCN3 and NEFL displayed the greatest differential expression by knocking down of SNAIL; the former was upregulated and the latter downregulated, respectively. Downregulation of CCN3 and upregulation of NEFL gene expression impaired the SNAIL-dependent EMT activity: high tumorigenicity, and chemoresistance in gastric cancer cells. Thus, approach that disrupts SNAIL/CCN3/NEFL axis may be credible in inhibiting gastric cancer development.
Assuntos
Carcinogênese/genética , Proteína Sobre-Expressa em Nefroblastoma/genética , Proteínas de Neurofilamentos/genética , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias Gástricas/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/genética , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Proteínas de Neurofilamentos/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição da Família Snail/genética , Estômago/patologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Objectives: An adequate development of the placenta includes trophoblast differentiation with the processes of trophoblast migration, invasion, cellular senescence and apoptosis which are all crucial to establishing a successful pregnancy. Altered placental development and function lead to placental diseases such as preeclampsia (PE) which is mainly characterized by insufficient trophoblast invasion and abnormally invasive placenta (AIP) disorders (Placenta accreta, increta, or percreta) which are characterized by excessive trophoblast invasion. Both of them will cause maternal and fetal morbidity/mortality. However, the etiology of these diseases is still unclear. Our previous study has shown that the matricellular protein nephroblastoma overexpressed (NOV, CCN3) induces G0/G1 cell cycle arrest, drives trophoblast cells into senescence and activates FAK and Akt kinases resulting in reduced cell proliferation and enhanced migration capability of the human trophoblast cell line SGHPL-5. The present study focuses on whether CCN3 can alter cell cycle-regulated pathways associated with trophoblast senescence and invasion activity in pathological versus gestational age-matched control placentas. Methods: Cell cycle regulator proteins were investigated by immunoblotting and qPCR. For localization of CCN3, p16, p21, and Cyclin D1 proteins, co-immunohistochemistry was performed. Results: In early-onset PE placentas, CCN3 was expressed at a significantly lower level compared to gestational age-matched controls. The decrease of CCN3 level is associated with an increase in p53, Cyclin E1 and pRb protein expression, whereas the level of cleaved Notch-1, p21, Cyclin D1, pFAK, pAKT, and pmTOR protein decreased. In term AIP placentas, the expression of CCN3 was significantly increased compared to matched term controls. This increase was correlated to an increase in p53, p16, p21, Cyclin D1, cleaved Notch-1, pFAK, pAkt, and pmTOR whereas pRb was significantly decreased. However, in late PE and early AIP placentas, no significant differences in CCN3, p16, p21, Cyclin D1, p53, and cleaved Notch-1 expression were found when matched to appropriate controls. Conclusions: CCN3 expression levels are correlated to markers of cell cycle arrest oppositely in PE and AIP by activating the FAK/AKT pathway in AIP or down-regulating in PE. This may be one mechanism to explain the different pathological features of placental diseases, PE and AIP.
Assuntos
Biomarcadores/metabolismo , Pontos de Checagem do Ciclo Celular , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Doenças Placentárias/patologia , Placenta/patologia , Pré-Eclâmpsia/patologia , Adulto , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Senescência Celular , Feminino , Humanos , Placenta/metabolismo , Doenças Placentárias/metabolismo , Pré-Eclâmpsia/metabolismo , GravidezRESUMO
BACKGROUND: Multiple sclerosis (MS) is an immune-mediated disease that damages myelin in the central nervous system (CNS). We investigated the profile of CCN3, a known regulator of immune function and a potential mediator of myelin regeneration, in multiple sclerosis in the context of disease state and disease-modifying treatment. METHODS: CCN3 expression was analysed in plasma, immune cells, CSF and brain tissue of MS patient groups and control subjects by ELISA, western blot, qPCR, histology and in situ hybridization. RESULTS: Plasma CCN3 levels were comparable between collective MS cohorts and controls but were significantly higher in progressive versus relapsing-remitting MS and between patients on interferon-ß versus natalizumab. Higher body mass index was associated with higher CCN3 levels in controls as reported previously, but this correlation was absent in MS patients. A significant positive correlation was found between CCN3 levels in matched plasma and CSF of MS patients which was absent in a comparator group of idiopathic intracranial hypertension patients. PBMCs and CD4+ T cells significantly upregulated CCN3 mRNA in MS patients versus controls. In the CNS, CCN3 was detected in neurons, astrocytes and blood vessels. Although overall levels of area immunoreactivity were comparable between non-affected, demyelinated and remyelinated tissue, the profile of expression varied dramatically. CONCLUSIONS: This investigation provides the first comprehensive profile of CCN3 expression in MS and provides rationale to determine if CCN3 contributes to neuroimmunological functions in the CNS.
Assuntos
Interferon beta/uso terapêutico , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/metabolismo , Natalizumab/uso terapêutico , Proteína Sobre-Expressa em Nefroblastoma/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Sobre-Expressa em Nefroblastoma/genética , Resultado do TratamentoRESUMO
Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of Ccn3 mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with p21 and p53 mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 in vitro, either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by H2O2 caused a dose-dependent increase in Ccn3 gene and CCN3 protein expression, along with enhanced expression of p21 and p53 mRNA and proteins, as well as SA-ß gal activity. Overexpression of CCN3 also enhanced p21 promoter activity via p53. Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of p21 and p53 mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.
Assuntos
Cartilagem Articular/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Osteoartrite do Joelho/metabolismo , Animais , Cartilagem Articular/crescimento & desenvolvimento , Linhagem Celular Tumoral , Células Cultivadas , Senescência Celular , Condrócitos/metabolismo , Condrócitos/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Sobre-Expressa em Nefroblastoma/genética , Osteoartrite do Joelho/patologia , Ratos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Systemic sclerosis (SSc) is a severe disease whose pathophysiology remains partly unknown, combining autoimmune, vascular, and fibrotic features. Recently, we evidenced a link between vasculopathy and pigmentary changes in SSc. CCN3 (NOV) is a matricellular protein implicated in both angiogenesis and pigmentation regulation, in particular melanocyte adhesion to the basal layer. We decided to study CCN3 expression in SSc epidermis. We show that in SSc patients with pigmentary changes compared to patients with normal pigmentation, CCN3 is specifically downregulated in situ in melanocytes and upregulated in keratinocytes. Moreover, the number of melanocytes is significantly decreased in SSc patients with a disease duration of more than 5 years compared to the other patients. Altogether, our findings could provide new insights on the mechanisms of pigmentary changes in SSc patients, as well as treatment adaptation in a personalized manner.