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
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
CCN3 is a matricellular protein that promotes oligodendrocyte progenitor cell differentiation and myelination in vitro and ex vivo. CCN3 is therefore a candidate of interest in central nervous system (CNS) myelination and remyelination, and we sought to investigate the expression and role of CCN3 during these processes. We found CCN3 to be expressed predominantly by neurons in distinct areas of the CNS, primarily the cerebral cortex, hippocampus, amygdala, suprachiasmatic nuclei, anterior olfactory nuclei, and spinal cord gray matter. CCN3 was transiently up-regulated following demyelination in the brain of cuprizone-fed mice and spinal cord lesions of mice injected with lysolecithin. However, CCN3-/- mice did not exhibit significantly different numbers of oligodendroglia or differentiated oligodendrocytes in the healthy or remyelinating CNS, compared to WT controls. These results suggest that despite robust and dynamic expression in the CNS, CCN3 is not required for efficient myelination or remyelination in the murine CNS in vivo.
Assuntos
Sistema Nervoso Central/metabolismo , Doenças Desmielinizantes/etiologia , Regulação da Expressão Gênica , Proteína Sobre-Expressa em Nefroblastoma/genética , Remielinização/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Imunofluorescência , Camundongos , Bainha de Mielina/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Oligodendroglia/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
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
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
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
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
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
Prostate cancer (PC) commonly metastasizes to the bone, resulting in pathologic fractures and poor prognosis. CCN3/nephroblastoma overexpressed is a secreted protein with a known role in promoting breast cancer metastasis to bone. However, in PC, CCN3 has been ascribed conflicting roles; some studies suggest that CCN3 promotes PC metastasis, whereas others argue a tumor suppressor role for CCN3 in this disease. Indeed, in the latter context, CCN3 has been shown to sequester the androgen receptor (AR) and suppress AR signaling. In the present study, we demonstrate that CCN3 functions as a bone-metastatic mediator, which is dependent on its C-terminal domain for this function. Analysis of tissue microarrays comprising >1500 primary PC patient radical prostatectomy specimens reveals that CCN3 expression correlates with aggressive disease and is negatively correlated with the expression of prostate-specific antigen, a marker of AR signaling. Together, these findings point to CCN3 as a biomarker to predict PC aggressiveness while providing clarity on its role as a functional mediator of PC bone metastasis.
Assuntos
Neoplasias Ósseas/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Calicreínas/biossíntese , Calicreínas/genética , Masculino , Metástase Neoplásica , Proteínas de Neoplasias , Antígeno Prostático Específico/biossíntese , Antígeno Prostático Específico/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/genéticaRESUMO
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
Osteoarthritis (OA), an age-related degenerative joint disease, is pathologically characterized by articular cartilage degeneration and synovial inflammation. Nephroblastoma overexpressed (NOV or CCN3), a matricellular protein, is a primary member of the CCN family (Cyr61, Ctgf, NOV) of proteins and is involved in various inflammatory disorders. Previous studies reported that CCN3 might play a therapeutic role in OA. However, the underlying mechanism remains unclear. In this study, we confirmed the expression of CCN3 was decreased in human and rat OA articular cartilage. Recombinant CCN3 ameliorated the IL-1ß-induced matrix catabolism, as demonstrated by MMP1, MMP3, MMP13, ADAMTS5 and iNOS expression, in vitro. In addition, the degradation of cartilage matrix such as collagen 2 and aggrecan could be reversed by CCN3. Furthermore, we found CCN3 promoted autophagy as Atg5, Beclin1 and LC3-II expression were increased. High-mobility group box 1 was negatively correlated with CCN3 in IL-1ß-induced osteoarthritis responses, and HMGB1 is involved in the protective effect of CCN3 in OA. Moreover, CCN3 overexpression decreased the expression of HMGB1 and reversed the IL-1ß induced MMPs production. Additionally, recombinant CCN3 or CCN3 overexpression attenuated the activation of PI3K/AKT/mTOR pathway induced by IL-1ß. Our study presents new mechanisms of CCN3 in osteoarthritis and indicates that CCN3 can serve as a novel potential therapeutic target for osteoarthritis.
Assuntos
Cartilagem Articular/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Osteoartrite/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Autofagia/fisiologia , Linhagem Celular Tumoral , Condrócitos/metabolismo , Humanos , Inflamação/metabolismo , Masculino , NF-kappa B/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were â¼20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Precursores de Proteínas/metabolismo , Animais , Células CHO , Linhagem Celular Tumoral , Fator de Crescimento do Tecido Conjuntivo/química , Cricetulus , Proteína Rica em Cisteína 61/química , Proteína Rica em Cisteína 61/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Camundongos , Proteína Sobre-Expressa em Nefroblastoma/química , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Domínios Proteicos , Precursores de Proteínas/química , Proteólise , Células RAW 264.7 , Ratos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: The liver microenvironment plays a key role in the progression and metastasis of hepatocellular carcinoma (HCC). Gene expression profiling of non-cancerous hepatic tissues obtained from patients with metastatic HCC exhibit a unique immune response signature, including upregulation of CCN3. However, the role of CCN3 secreted from non-cancerous hepatic tissues in the progression of HCC remains unclear. METHODS: Using tissue microarrays, we examined CCN3 in non-cancerous hepatic tissues of patients with HCC and correlated expression with clinical and pathological features. In addition, CCN3 localization and mechanisms of HCC progression were investigated in tissues and cell lines. Finally, correlations between CCN3 and cirrhosis were explored in patients. RESULTS: CCN3 was primarily localized to hepatic cells of non-cancerous hepatic tissues and was associated with vascular invasion and poor prognosis in patients with HCC. CCN3 expression in non-cancerous hepatic tissues also correlated with the degree of liver fibrosis. Compared with conditioned media from wild-type LO2 cells, conditioned media from hepatic cell line LO2 activated by LX2 (aLO2-CM) induced CCN3 expression and HCC cell proliferation and metastasis. Further, aLO2-CM activated MAPK signaling and epithelial-mesenchymal transition in HCC cells. Finally, CCN3 was inversely related to cirrhosis in the prognosis of HCC and negatively regulated hepatic stellate cells (HSCs) in vitro with downregulation of α-SMA, TGF-ß, and collagens. CONCLUSIONS: CCN3 was secreted from hepatic cells activated by HSCs and increased MAPK signaling, EMT, proliferation and metastasis of HCC cells. CCN3 was also inversely related to cirrhosis, regulating HSCs through a negative feedback loop.
Assuntos
Carcinoma Hepatocelular/genética , Hepatócitos/metabolismo , Neoplasias Hepáticas/genética , Proteína Sobre-Expressa em Nefroblastoma/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hepatócitos/patologia , Humanos , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Comunicação Parácrina/genética , Transdução de Sinais/genética , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: Hepatic stellate cells (HSCs) have a key role in fibrogenesis and in the filtrates of the hepatocellular carcinoma (HCC) stroma, in which they are remodeled and play a critical role in HCC progression. However, the precise role of HSCs trending, infiltration and paracrine in orchestrating the stroma-derived oxaliplatin-resistance in HCC is still vague. METHODS: The chemo-resistant models were established to explore the correlation between HSC cells and the condition of chemoresistance. The HCC clinical samples were collected to confirm this phenomenon. Then, the relationship between secretory CCN3 from oxaliplatin-resistant HCC and the infiltration of HSCs in associated HCC microenvironment was evaluated. Finally, the role and mechanism of HSCs remodeling in the orchestration of oxaliplatin-resistant HCC were explored. RESULTS: The increased infiltration of HSCs and collagen accumulation were found in the microenvironment of oxaliplatin-resistant HCC. The cDNA profiles of the oxaliplatin-resistant HCC was reanalyzed, and CCN3 was one of the significantly increased genes. In HCC clinical samples, the levels of CCN3 and α-SMA are positively correlated, and high expression of CCN3 and α-SMA are positively associated with malignant phenotype and poor prognosis. Then the enhanced abilities of migration and proliferation of HSCs, and elevation of the cytokines paracrine from HSCs relating to HCC malignancy were proved in vitro and in vivo, and which were related to CCN3-ERK signaling pathway activation. CONCLUSIONS: HSCs remodeling are positively related to CCN3 paracrine in hepatocellular carcinoma, which orchestrated the stroma-derived resistance to chemotherapy in HCC.
Assuntos
Carcinoma Hepatocelular/patologia , Resistencia a Medicamentos Antineoplásicos , Células Estreladas do Fígado/patologia , Neoplasias Hepáticas/patologia , Proteína Sobre-Expressa em Nefroblastoma/genética , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Actinas/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colágeno/metabolismo , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Transplante de Neoplasias , Oxaliplatina , Comunicação Parácrina , Prognóstico , Microambiente Tumoral , Regulação para CimaRESUMO
BACKGROUND Matricellular proteins of the extracellular matrix (ECM) include tenascin-C (TNC) and cellular communication network factor 3 (CCN3). This study aimed to investigate the role of TNC and CCN3 as prognostic factors for post hepatectomy liver failure (PHLF) in a rat model of partial hepatectomy and 50 patients following partial hepatectomy. MATERIAL AND METHODS Sprague-Dawley rats underwent 85% (n=53) or 90% hepatectomy (n=53) in the partial hepatectomy (PHx) model. TNC and CCN3 mRNA expression in residual liver tissue was evaluated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and enzyme-linked immunoassay (ELISA) determined the serum levels of TNC and CCN3. In 50 patients who underwent partial hepatectomy, TNC and CCN3 serum levels were measured on postoperative day 1 and day 3. RESULTS In the rat partial hepatectomy model, mRNA and serum levels of TNC and CCN3 were significantly increased within the first 24 h, and were higher in the 90% PHx group compared with the 85% PHx group. Fifty patients who underwent partial hepatectomy, included patients with PHLF (n=12) and patients without PHLF (n=38). Multivariate analysis confirmed that serum levels on postoperative day 3 TNChigh+CCN3high was a significant predictor of PHLF, which was associated with more than twice the risk of severe morbidity when compared with the low-risk patients (80% vs. 30%) and a significantly longer hospital stay (17 days vs. 8 days). CONCLUSIONS Further studies are needed to evaluate the potential role of the matricellular proteins, TNC and CCN3 as early clinical predictors for PHLF.
Assuntos
Hepatectomia/efeitos adversos , Falência Hepática/etiologia , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Tenascina/metabolismo , Adulto , Idoso , Animais , Área Sob a Curva , Bilirrubina/sangue , Modelos Animais de Doenças , Feminino , Humanos , Tempo de Internação , Falência Hepática/sangue , Falência Hepática/genética , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Morbidade , Análise Multivariada , Proteína Sobre-Expressa em Nefroblastoma/sangue , Proteína Sobre-Expressa em Nefroblastoma/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Curva ROC , Ratos Sprague-Dawley , Fatores de Risco , Análise de Sobrevida , Tenascina/sangue , Tenascina/genéticaRESUMO
Bone morphogenetic proteins (BMPs) are secreted growth factors that promote differentiation processes in embryogenesis and tissue development. Regulation of BMP signaling involves binding to a variety of extracellular proteins, among which are many von Willebrand factor C (vWC) domain-containing proteins. Although the crystal structure of the complex of crossveinless-2 (CV-2) vWC1 and BMP-2 previously revealed one mode of the vWC/BMP-binding mechanism, other vWC domains may bind to BMP differently. Here, using X-ray crystallography, we present for the first time structures of the vWC domains of two proteins thought to interact with BMP-2: collagen IIA and matricellular protein CCN3. We found that these two vWC domains share a similar N-terminal fold that differs greatly from that in CV-2 vWC, which comprises its BMP-2-binding site. We analyzed the ability of these vWC domains to directly bind to BMP-2 and detected an interaction only between the collagen IIa vWC and BMP-2. Guided by the collagen IIa vWC domain crystal structure and conservation of surface residues among orthologous domains, we mapped the BMP-binding epitope on the subdomain 1 of the vWC domain. This binding site is different from that previously observed in the complex between CV-2 vWC and BMP-2, revealing an alternative mode of interaction between vWC domains and BMPs.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Colágeno/química , Colágeno/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/química , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Fator de von Willebrand/química , Sítios de Ligação , Proteína Morfogenética Óssea 2/química , Células Cultivadas , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Fator de von Willebrand/metabolismoRESUMO
The ductus arteriosus (DA), an essential fetal shunt between the pulmonary trunk and the descending aorta, changes its structure during development. Our previous studies have demonstrated that prostaglandin E2 (PGE2)-EP4 signaling promotes intimal cushion formation (ICF) by activating the migration of DA smooth muscle cells via the secretion of hyaluronan. We hypothesized that, in addition to hyaluronan, PGE2 may secrete other proteins that also regulate vascular remodeling in the DA. In order to detect PGE2 stimulation-secreted proteins, we found that CCN3 protein was increased in the culture supernatant in the presence of PGE2 in a dose-dependent manner by nano-flow liquid chromatography coupled with tandem mass spectrometry analysis and enzyme-linked immunosorbent assay. Quantitative RT-PCR analysis revealed that PGE2 stimulation tended to increase the expression levels of CCN3 mRNA in DA smooth muscle cells. Immunohistochemical analysis revealed that CCN3 was highly localized in the entire smooth muscle layers and the endothelium of the DA. Furthermore, exogenous CCN3 inhibited PGE2-induced ICF in the ex vivo DA tissues. These results suggest that CCN3 is a secreted protein of the DA smooth muscle cells induced by PGE2 to suppress ICF of the DA. The present study indicates that CCN3 could be a novel negative regulator of ICF in the DA to fine-tune the PGE2-mediated DA remodeling.
Assuntos
Dinoprostona/metabolismo , Canal Arterial/embriologia , Ácido Hialurônico/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Ratos Wistar/embriologia , Animais , Movimento Celular , Células Cultivadas , Canal Arterial/citologia , Canal Arterial/metabolismo , Miócitos de Músculo Liso/citologia , Técnicas de Cultura de Órgãos , Ratos Wistar/metabolismo , Remodelação VascularRESUMO
Normal extracellular secretion of nephroblastoma overexpressed (NOV, also known as CCN3) is important for the adhesion, migration, and differentiation of cells. In previous studies, we have shown that the intracellular accumulation of CCN3 inhibits the growth of prominent neurons. Increased intracellular CCN3 can be induced through various processes, such as transcription, detoxification, and posttranslational modification. In general, posttranslational modifications are very important for protein secretion. However, it is unclear whether posttranslational modification is necessary for CCN3 secretion. In this study, we have conducted mutational analysis of CCN3 to demonstrate that its thrombospondin type-1 (TSP1) domain is important for CCN3 secretion and intracellular function. Point mutation analysis confirmed that CCN3 secretion was inhibited by cysteine (C)241 mutation, and overexpression of CCN3-C241A inhibited neuronal axonal growth in vivo. Furthermore, we demonstrated that palmitoylation is important for the extracellular secretion of CCN3 and that zinc finger DHHC-type containing 22 (ZDHHC22), a palmityoltransferase, can interact with CCN3. Taken together, our results suggest that palmitoylation by ZDHHC22 at C241 in the CCN3 TSP1 domain may be required for the secretion of CCN3. Aberrant palmitoylation induces intracellular accumulation of CCN3, inhibiting neuronal axon growth.
Assuntos
Carnitina O-Palmitoiltransferase/química , Carnitina O-Palmitoiltransferase/metabolismo , Lipoilação/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/química , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Neurônios/metabolismo , Animais , Sítios de Ligação , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Neurônios/química , Neurônios/citologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Growth factors like TGFß and CTGF (CCN2) plays a vital role in various cellular functions. TGFß and CTGF are overexpressed in renal fibrosis. CTGF act as profibrotic stimuli to TGFß. CCN3 is a member of CCN family which also comprises CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2) and CCN6 (WISP-3). CCN3 has been shown to antagonise CTGF. In this study, we investigated the role of CCN3 in TGFß1-mediated signalling in human podocytes culture. This study describes the novel function of CCN3 in regulation of TGFß1 mediated non-canonical Smad signalling in human podocytes culture. Experiments were conducted on conditionally immortalised human podocytes incubated with TGFß1 (1.25ng/ml and 2.5ng/ml) and CCN3 (360ng/ml). Western blot study was performed to study signalling proteins. RT-PCR was performed to study alternative splicing of Fibronectin (Fn). Real time PCR was performed to look for gene expression of Fn and collagen IV and collagen I. TGFß1 induced the Smad1/5/8, Smad3 and p38 phosphorylation and CCN3 downregulated the TGFß1 induced Smad1/5/8 phosphorylation and did not affect Smad3 and p38 phosphorylation. In addition to this CCN3 induced alternative splicing of Extra domain A Fibronectin (EDA+Fn). CCN3 also induced collagen IV, Collagen I and Fn gene expression. This is the first evidence of downregulation of TGFß-mediated activation of a Smad1/5/8 signalling pathway by CCN3 in human podocytes and in any cell type. Targeting CCN3-mediated events could provide exciting outcomes in the understanding of molecular mechanism of fibrosis.