RESUMEN
Atrial fibrillation (AF) is associated with oxidative stress and Ca2+-handling abnormalities in atrial myocytes. Our prior study has demonstrated the involvement of CD44, a membrane receptor for hyaluronan (HA), in the pathogenesis of AF. This study further evaluated whether CD44 and its related signaling mediate atrial tachycardia-induced oxidative stress and Ca2+-handling abnormalities. Tachypacing in atrium-derived myocytes (HL-1 cell line) induced the activation of CD44-related signaling, including HA and HA synthase (HAS) expression. Blocking HAS/HA/CD44 signaling attenuated tachypacing-induced oxidative stress (NADPH oxidase [NOX] 2/4 expression) and Ca2+-handling abnormalities (oxidized Ca2+/calmodulin-dependent protein kinase II [ox-CaMKII] and phospho-ryanodine receptor type 2 [p-RyR2] expression) in HL-1 myocytes. Furthermore, a direct association between CD44 and NOX4 was documented in tachy-paced HL-1 myocytes and atrial tissues from AF patients. In vitro, Ca2+ spark frequencies in atrial myocytes isolated from CD44-/- mice were lower than those from wild-type mice. Furthermore, administration of an anti-CD44 blocking antibody in atrial myocytes isolated from wild-type mice diminished the frequency of Ca2+ spark. Ex vivo tachypacing models of CD44-/- mice exhibited a lower degree of oxidative stress and expression of ox-CaMKII/p-RyR2 in their atria than those of wild-type mice. In vivo, burst atrial pacing stimulated a less inducibility of AF in CD44-/-mice than in wild-type mice. In conclusion, atrial tachypacing-induced Ca2+-handling abnormalities are mediated via CD44/NOX4 signaling, which provides a possible explanation for the development of AF.
Asunto(s)
Fibrilación Atrial/genética , Remodelación Atrial/genética , Atrios Cardíacos/metabolismo , NADPH Oxidasa 4/genética , Taquicardia/genética , Animales , Fibrilación Atrial/metabolismo , Fibrilación Atrial/patología , Remodelación Atrial/fisiología , Señalización del Calcio/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Atrios Cardíacos/patología , Humanos , Receptores de Hialuranos/genética , Ácido Hialurónico/genética , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , NADPH Oxidasa 2/genética , Canal Liberador de Calcio Receptor de Rianodina , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/patología , Transducción de Señal/genética , Taquicardia/patologíaRESUMEN
Atrial fibrillation (AF) is associated with atrial fibrosis. Inhibition of atrial fibrosis might be a plausible approach for AF prevention and therapy. This study is designed to evaluate the potential role of CD44, a membrane receptor known to regulate fibrosis, and its related signaling in the pathogenesis of atrial fibrosis and AF. Treatment of cultured rat atrial fibroblasts with transforming growth factor-ß (TGF-ß, a key mediator of atrial fibrosis) led to a higher expression of hyaluronan (HA), CD44, STAT3, and collagen (a principal marker of fibrosis) than that of ventricular fibroblasts. In vivo, TGF-ß transgenic mice and AF patients exhibited a greater expression of HA, CD44, STAT3, and collagen in their atria than wild-type mice and sinus rhythm subjects, respectively. Treating TGF-ß transgenic mice with an anti-CD44 blocking antibody resulted in a lower expression of STAT3 and collagen in their atria than those with control IgG antibody. Programmed stimulation triggered less AF episodes in TGF-ß transgenic mice treated with anti-CD44 blocking antibody than in those with control IgG. Blocking CD44 signaling with anti-CD44 antibody and mutated CD44 plasmids attenuated TGF-ß-induced STAT3 activation and collagen expression in cultured atrial fibroblasts. Deletion and mutational analysis of the collagen promoter along with chromatin immunoprecipitation demonstrated that STAT3 served as a vital transcription factor in collagen expression. TGF-ß-mediated HA/CD44/STAT3 pathway plays a crucial role in the development of atrial fibrosis and AF. Blocking CD44-dependent signaling may be a feasible way for AF management.
Asunto(s)
Fibrilación Atrial/metabolismo , Remodelación Atrial , Atrios Cardíacos/metabolismo , Receptores de Hialuranos/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Fibrilación Atrial/patología , Fibrilación Atrial/fisiopatología , Fibrilación Atrial/prevención & control , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Atrios Cardíacos/patología , Atrios Cardíacos/fisiopatología , Frecuencia Cardíaca , Receptores de Hialuranos/genética , Ácido Hialurónico/metabolismo , Masculino , Ratones Endogámicos DBA , Ratones Transgénicos , Ratas Wistar , Factor de Transcripción STAT3/genética , Transducción de Señal , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Cancer progression is commonly segregated into processes of primary tumour growth and secondary metastasis. Recent evidence suggests that a subpopulation of cancer cells, cancer stem cells (CSCs), is responsible for tumour growth in cancer. However, the role of CSCs in cancer metastasis is unclear. In this study, we found that the C terminus of CD44 contributes to sphere formation and survival in vitro via the CD44-SRC-integrin axis. In addition, nuclear CD44/acetylated-STAT3 is required for clonal formation in vitro and tumourigenicity in vivo. Nuclear CD44 binds to various promoters identified by chromatin immunoprecipitation-seq, including that of c-myc and Twist1, leading to cell fate change through transcriptional reprogramming. We propose that nuclear CD44/acetylated-STAT3 performs an unexpected tumour-progressing function by enhancing cell outgrowth into structures where cells with properties of CSCs can be generated from differentiated somatic cells in suspension culture, and then exhibit attributes of cells that have undergone an epithelial-mesenchymal transition, leading to tumour metastasis, and a resulting worse prognosis.
Asunto(s)
Neoplasias del Colon/patología , Receptores de Hialuranos/metabolismo , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Inmunoprecipitación de Cromatina , ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , Regiones Promotoras Genéticas , Unión Proteica , Transducción de Señal , Transcripción Genética , Familia-src Quinasas/metabolismoAsunto(s)
Terapia Electroconvulsiva/tendencias , Consentimiento Informado/legislación & jurisprudencia , Programas Obligatorios/tendencias , Auditoría Médica/estadística & datos numéricos , Competencia Mental/legislación & jurisprudencia , Terapia Electroconvulsiva/estadística & datos numéricos , Humanos , Programas Obligatorios/estadística & datos numéricos , VictoriaRESUMEN
BACKGROUND: Atrial fibrillation (AF), a common arrhythmia in clinics, is characterized as downregulation of L-type calcium channel (LTCC) and shortening of atrial action potential duration (APD). Our prior studies have shown the association of CD44 with AF genesis. OBJECTIVE: The purpose of this study was to explore the potential role of CD44 and its related signaling in tachypacing-induced downregulation of LTCC. METHODS AND RESULTS: In vitro, tachypacing in atrium-derived myocytes (HL-1 cell line) induced activation (phosphorylation) of cyclic adenosine monophosphate response element-binding protein (CREB). Furthermore, tachypacing promoted an association between CREB and CD44 in HL-1 myocytes, which was documented in atrial tissues from patients with AF. Deletion and mutational analysis of the LTCC promoter along with chromatin immunoprecipitation revealed that cyclic adenosine monophosphate response element is essential for tachypacing-inhibited LTCC transcription. Tachypacing also hindered the binding of p-CREB to the promoter of LTCC. Blockade of CREB/CD44 signaling in HL-1 cells attenuated tachypacing-triggered downregulation of LTCC and shortening of APD. Atrial myocytes isolated from CD44-/- mice exhibited higher LTCC current and longer APD than did those from wild-type mice. Ex vivo, tachypacing caused less activation of CREB in CD44-/- mice than in wild-type mice. In vivo, burst atrial pacing stimulated less inducibility of AF in CREB inhibitor-treated mice than in controls. CONCLUSION: Tachypacing-induced CREB/CD44 signaling contributes to the suppression of LTCC, which provides valuable information about the pathogenesis of atrial modeling and AF.
Asunto(s)
Fibrilación Atrial/terapia , Remodelación Atrial/fisiología , Proteína de Unión a CREB/genética , Canales de Calcio Tipo L/genética , Estimulación Cardíaca Artificial/métodos , Regulación de la Expresión Génica , Receptores de Hialuranos/genética , Animales , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Western Blotting , Proteína de Unión a CREB/biosíntesis , Canales de Calcio Tipo L/biosíntesis , Línea Celular , ADN/genética , Modelos Animales de Enfermedad , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Atrios Cardíacos/fisiopatología , Receptores de Hialuranos/biosíntesis , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Transducción de SeñalRESUMEN
Epithelial-mesenchymal transition (EMT)/mesenchymal-epithelial transition (MET) processes are proposed to be a driving force of cancer metastasis. By studying metastasis in bone marrow-derived mesenchymal stem cell (BM-MSC)-driven lung cancer models, microarray time-series data analysis by systems biology approaches revealed BM-MSC-induced signaling triggers early dissemination of CD133+/CD83+ cancer stem cells (CSCs) from primary sites shortly after STAT3 activation but promotes proliferation towards secondary sites. The switch from migration to proliferation was regulated by BM-MSC-secreted LIF and activated LIFR/p-ERK/pS727-STAT3 signaling to promote early disseminated cancer cells MET and premetastatic niche formation. Then, tumor-tropic BM-MSCs circulated to primary sites and triggered CD151+/CD38+ cells acquiring EMT-associated CSC properties through IL6R/pY705-STAT3 signaling to promote tumor initiation and were also attracted by and migrated towards the premetastatic niche. In summary, STAT3 phosphorylation at tyrosine 705 and serine 727 differentially regulates the EMT-MET switch within the distinct molecular subtypes of CSCs to complete the metastatic process.
Asunto(s)
Transición Epitelial-Mesenquimal , Metástasis de la Neoplasia , Factor de Transcripción STAT3/metabolismo , Línea Celular Tumoral , Humanos , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia/fisiología , Neoplasias Pulmonares/patología , Sistema de Señalización de MAP Quinasas/fisiología , Células Madre Mesenquimatosas/fisiología , Células Madre Neoplásicas/fisiología , Fosforilación , Mapas de Interacción de Proteínas , Receptores de Interleucina-6/fisiología , Serina , TirosinaRESUMEN
BACKGROUND AND AIMS: Uremic patients are characterized by an increased risk of atherosclerotic cardiovascular diseases. Vascular smooth muscle cell (VSMC) proliferation contributes to neointimal formation, a main pathological feature in atherosclerosis. Activation of CREB/ATF3 signaling is pivotal in VSMC proliferation, yet its role in uremic atherosclerosis is unknown. This study aimed to explore whether CREB/ATF3 signaling is involved in the molecular mechanism underlying neointimal formation in uremia. METHODS AND RESULTS: Treatment of VSMCs with uremic toxin (indoxyl sulfate [IS]) activated cAMP/CREB/ATF3/cyclin D signaling, which was reflected by increased VSMC proliferation. Blocking cAMP/PKA/CREB/ATF3 signaling attenuated the promoting effect of IS on cyclin D1 expression and VSMC proliferation. Loss-of-function and time-dependent experiments showed that ATF3 lies downstream of the CREB signaling. Mutational analysis of cyclin D1 promoter along with chromatin immunoprecipitation assays showed that CREB/ATF3 signaling participated in IS-induced cyclin D transcription. In vivo, phosphorylated CREB (an active form of CREB) and ATF3 were prominently upregulated in the neointima of experimental uremic rats, the atherosclerotic plaques of uremic ApoE-/- mice, and the iliac arteries of uremic patients. Notably, the use of lentivirus to knock down ATF3 in the neointima of balloon-injured arteries could suppress the effect of uremia in vivo, including neointimal formation and cyclin D expression. CONCLUSIONS: In this study, we demonstrated that CREB/ATF3-related signaling may be involved in IS-induced VSMC proliferation and the pathogenesis of neointimal formation during uremia.
Asunto(s)
Neointima , Uremia , Factor de Transcripción Activador 3/genética , Animales , Proliferación Celular , Células Cultivadas , Humanos , Indicán , Ratones , Músculo Liso Vascular , Miocitos del Músculo Liso , RatasRESUMEN
Osteopontin (OPN) and splice variants of CD44 (CD44(V)) have independently been identified as markers for tumor progression. In this study, we show that both OPN and CD44(V) are frequently overexpressed in human gastric cancer and that OPN-engaged CD44(V) ligation confers cells an increased survival mediated through integrin activation. First, we show that OPN treatment confers cells an increased resistance to UV-induced apoptosis. The OPN-mediated antiapoptosis is dependent on the expression of the variant exon 6 (V6)- or V7-containing CD44 as shown by overexpression of individual CD44(V) in gastric AZ521 cells that express no or very low level of endogenous CD44 and by knockdown of the constitutively expressed V6-containing CD44 isoforms in colon HT29 cells. Although OPN also interacts with RGD integrins, OPN-RGD sequence is dispensable for OPN-mediated antiapoptosis. OPN-induced antiapoptosis is mainly attributed to the engagement of CD44(V) isoforms and the relay of an inside-out signaling via Src activity, leading to robust integrin activation. Furthermore, OPN-elicited antiapoptosis was observed when cells were plated on fibronectin but not on poly-D-lysin, and preincubation of cells with anti-integrin beta(1) antibody to block integrin-extracellular matrix (ECM) interaction or ectopic expression of the dominant-negative forms of focal adhesion kinase to block ECM-derived signal abolished OPN-induced survival, suggesting that OPN-elicited antiapoptotic function is propagated from matrix transduced by integrin. Taken together, we showed that OPN-CD44(V) interaction promotes ECM-derived survival signal mediated through integrin activation, which may play an important role in the pathogenic development and progression of gastric cancer.
Asunto(s)
Adenocarcinoma/patología , Neoplasias Gastrointestinales/patología , Receptores de Hialuranos/metabolismo , Integrinas/metabolismo , Osteopontina/fisiología , Adenocarcinoma/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Gastrointestinales/metabolismo , Células HT29 , Humanos , Osteopontina/metabolismo , Osteopontina/farmacología , Isoformas de Proteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Rayos Ultravioleta/efectos adversosRESUMEN
Secreted frizzled-related proteins (SFRPs) are mainly known for their role as extracellular modulators and tumor suppressors that downregulate Wnt signaling. Using the established (CRISPR/Cas9 targeting promoters of SFRPs and targeting SFRPs transcript) system, we find that nuclear SFRPs interact with ß-catenin and either promote or suppress TCF4 recruitment. SFRPs bind with ß-catenin on both their N and C termini, which the repressive effects caused by SFRP-ß-catenin-N-terminus binding overpower the promoting effects of their binding at the C terminus. By high Wnt activity, ß-catenin and SFRPs only bind with their C termini, which results in the upregulation of ß-catenin transcriptional activity and cancer stem cell (CSC)-related genes. Furthermore, we identify disulfide bonds of the cysteine-rich domain (CRD) and two threonine phosphorylation events of the netrin-related motif (NTR) domain of SFRPs that are essential for their role as biphasic modulators, suggesting that SFRPs are biphasic modulators of Wnt signaling-elicited CSC properties beyond extracellular control.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Vía de Señalización Wnt/genética , HumanosRESUMEN
CD133 is widely used as a surface marker to isolate cancer stem cells (CSCs). Here we show that in CSCs CD133 contributes to ß-catenin-mediated transcriptional activation and to the self-renewal capacity of sphere-forming and side-population (SP) cells in cell lines from brain, colon and lung cancers, but not gastric or breast cancers. In chromatin immunoprecipitation assays, ß-catenin binding to the proximal promoter regions of ITGA2-4 and ITGA10-11 in brain, colon and lung cancer cell lines could be triggered by CD133, and ß-catenin also bound to the proximal promoter regions of ITGB6 and ITGB8 in cell lines from gastric and breast cancers. CD133 thus induces ß-catenin binding and transcriptional activation of diverse targets that are cancer type-specific. Cell migration triggered by wounding CD133+ cells cultured on ECM-coated dishes can induce polarity and lipid raft coalescence, enhancing CD133/integrin signaling and asymmetric cell division. In response to directional cues, integrins, Src and the Par complex were enriched in lipid rafts, and the assembly and activation of an integrated CD133-integrin-Par signaling complex was followed by Src/Akt/GSK3ß signaling. The subsequent increase and nuclear translocation of ß-catenin may be a regulatory switch to increase drug resistance and stemness properties. Collectively, these findings 1) indicate that a polarized cell migration-induced CD133/integrin/Src/Akt/GSK3ß/ß-catenin axis is required for maintenance of CSC properties, 2) establish a function for CD133 and 3) support the rationale for targeting CD133 in cancer treatment.
Asunto(s)
Antígenos CD/metabolismo , Movimiento Celular , Glucógeno Sintasa Quinasa 3/metabolismo , Glicoproteínas/metabolismo , Integrinas/metabolismo , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/patología , Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , beta Catenina/metabolismo , Antígeno AC133 , Animales , Apoptosis , Biomarcadores de Tumor/metabolismo , Western Blotting , Adhesión Celular , Proliferación Celular , Inmunoprecipitación de Cromatina , Femenino , Glucógeno Sintasa Quinasa 3 beta , Humanos , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/metabolismo , Ratones , Ratones SCID , Células Madre Neoplásicas/metabolismo , Células de Población Lateral/metabolismo , Células de Población Lateral/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Wnt signaling contributes to the reprogramming and maintenance of cancer stem cell (CSC) states that are activated by epithelial-mesenchymal transition (EMT). However, the mechanistic relationship between EMT and the Wnt pathway in CSC is not entirely clear. Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) indicated that EMT induces a switch from the ß-catenin/E-cadherin/Sox15 complex to the ß-catenin/Twist1/TCF4 complex, the latter of which then binds to CSC-related gene promoters. Tandem coimmunoprecipitation and re-ChIP experiments with epithelial-type cells further revealed that Sox15 associates with the ß-catenin/E-cadherin complex, which then binds to the proximal promoter region of CASP3. Through this mechanism, Twist1 cleavage is triggered to regulate a ß-catenin-elicited promotion of the CSC phenotype. During EMT, we documented that Twist1 binding to ß-catenin enhanced the transcriptional activity of the ß-catenin/TCF4 complex, including by binding to the proximal promoter region of ABCG2, a CSC marker. In terms of clinical application, our definition of a five-gene CSC signature (nuclear ß-catenin(High)/nuclear Twist1(High)/E-cadherin(Low)/Sox15(Low)/CD133(High)) may provide a useful prognostic marker for human lung cancer.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Neoplasias Pulmonares/genética , Células Madre Neoplásicas/metabolismo , beta Catenina/genética , Antígeno AC133 , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Western Blotting , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones SCID , Recurrencia Local de Neoplasia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Factores de Transcripción SOX/genética , Factores de Transcripción SOX/metabolismo , Trasplante Heterólogo , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
Canine mammary tumors (CMTs) have been proposed to be a good animal model for human breast cancer. To provide a basis for the tumorigenic study of CMTs, cell lines were established using a modified cell culture technique. The epithelial morphology and immunostaining with cytokeratin 18 confirmed the epithelial origin of the cells. In an investigation of possible mammary tumorigenesis-related factors, the expression of Wnt signaling-related proteins was detected in cell lines. Secreted frizzled-related protein 2 (SFRP2) was abundantly expressed in CMT cells but not in normal canine mammary gland (MG) cells. Secreted frizzled-related protein 2 was secreted into the culture medium and was associated with the extracellular matrix. In addition, increased expressions of beta-catenin and cyclin D1 were observed in cells overexpressing SFRP2. The marked differential expression of SFRP2 reveals that this protein may be a potential candidate marker for CMTs. The CMT cell line established in this study provides a useful tool and experimental model for understanding both the tumorigenesis of CMTs and the role of Wnt signaling in cancers.
Asunto(s)
Neoplasias de la Mama , Técnicas de Cultivo de Célula/métodos , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/citología , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular Tumoral , Ciclina D1/metabolismo , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Perros , Matriz Extracelular/metabolismo , Femenino , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Queratinas , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/metabolismo , Proteínas Wnt , beta CateninaRESUMEN
Neurite outgrowth is an essential process during neuronal differentiation as well as neuroregeneration. Thus, understanding the molecular and cellular control of neurite outgrowth will benefit patients with neurological diseases. We have previously shown that overexpression of the signaling adaptor protein SH2B1ß promotes fibroblast growth factor 1 (FGF1)-induced neurite outgrowth (W. F. Lin, C. J. Chen, Y. J. Chang, S. L. Chen, I. M. Chiu, and L. Chen, Cell. Signal. 21:1060-1072, 2009). SH2B1ß also undergoes nucleocytoplasmic shuttling and regulates a subset of neurotrophin-induced genes. Although these findings suggest that SH2B1ß regulates gene expression, the nuclear role of SH2B1ß was not known. In this study, we show that SH2B1ß interacts with the transcription factor, signal transducer, and activator of transcription 3 (STAT3) in neuronal PC12 cells, cortical neurons, and COS7 fibroblasts. By affecting the subcellular distribution of STAT3, SH2B1ß increased serine phosphorylation and the concomitant transcriptional activity of STAT3. As a result, overexpressing SH2B1ß enhanced FGF1-induced expression of STAT3 target genes Egr1 and Cdh2. Chromatin immunoprecipitation assays further reveal that, in response to FGF1, overexpression of SH2B1ß promotes the in vivo occupancy of STAT3-Sp1 heterodimers at the promoter of Egr1 and Cdh2. These findings establish a central role of SH2B1ß in orchestrating signaling events to transcriptional activation through interacting and regulating STAT3-containing complexes during neuronal differentiation.
Asunto(s)
Proteínas Portadoras/metabolismo , Diferenciación Celular/genética , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Expresión Génica/genética , Neuronas/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Células COS , Cadherinas/genética , Cadherinas/metabolismo , Proteínas Portadoras/genética , Línea Celular Tumoral , Chlorocebus aethiops , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Factor 1 de Crecimiento de Fibroblastos/genética , Células HEK293 , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células PC12 , Fosforilación/genética , Regiones Promotoras Genéticas/genética , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/genéticaRESUMEN
BACKGROUND: Inherited cardiac conduction diseases (CCD) are rare but are caused by mutations in a myriad of genes. Recently, whole-exome sequencing has successfully led to the identification of causal mutations for rare monogenic Mendelian diseases. OBJECTIVE: To investigate the genetic background of a family affected by inherited CCD. METHODS AND RESULTS: We used whole-exome sequencing to study a Chinese family with multiple family members affected by CCD. Using the pedigree information, we proposed a heterozygous missense mutation (c.G695T, Gly232Val) in the lamin A/C (LMNA) gene as a candidate mutation for susceptibility to CCD in this family. The mutation is novel and is expected to affect the conformation of the coiled-coil rod domain of LMNA according to a structural model prediction. Its pathogenicity in lamina instability was further verified by expressing the mutation in a cellular model. CONCLUSIONS: Our results suggest that whole-exome sequencing is a feasible approach to identifying the candidate genes underlying inherited conduction diseases.
Asunto(s)
Arritmias Cardíacas/genética , Exoma , Enfermedades Genéticas Congénitas/genética , Sistema de Conducción Cardíaco/anomalías , Lamina Tipo A/genética , Mutación Missense , Linaje , Adulto , Anciano , Anciano de 80 o más Años , Sustitución de Aminoácidos , Síndrome de Brugada , Trastorno del Sistema de Conducción Cardíaco , Análisis Mutacional de ADN , Familia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estructura Terciaria de ProteínaRESUMEN
Expression of the type I transmembrane glycoprotein CD44 has recently been recognized as a signature for cancer stem cells. In this study, we demonstrate that CD44, once engaged, is internalized and translocated to the nucleus, where it binds to various promoters, including that of cyclin D1, leading to cell fate change through transcriptional reprogramming. In regulating cyclin D1 expression, the internalized CD44 forms a complex with STAT3 and p300 (acetyltransferase), eliciting STAT3 acetylation at lysine 685 and dimer formation in a cytokine- and growth factor-independent manner. A bipartite nuclear localization signal (NLS) was mapped to the cytoplasmic tail of CD44, which mediates its nuclear translocation. Expression of CD44(NLS) mutant sequesters STAT3 in cytosol. In the nucleus, the acetylated STAT3 dimer remains associated with CD44 and binds to the cyclin D1 promoter, leading to increased cyclin D1 expression and cell proliferation. This study describes a novel function for CD44 in transcriptional modulation through nuclear translocation of the internalized CD44 and complex formation with transcription factors.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Receptores de Hialuranos/metabolismo , Factor de Transcripción STAT3/metabolismo , Acetilación , Ciclo Celular/fisiología , Línea Celular , Proliferación Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Endosomas/metabolismo , Mucosa Gástrica/citología , Mucosa Gástrica/metabolismo , Humanos , Señales de Localización Nuclear , Factor de Transcripción STAT3/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
CD44 is present in detergent-resistant, cholesterol-rich microdomains, called lipid rafts, in many types of cells. However, the functional significance of CD44 in lipid rafts is still unknown. We have previously demonstrated that osteopontin-mediated engagement of CD44 spliced variant isoforms promotes an extracellular matrix-derived survival signal through integrin activation. By using a series of CD44 mutants and pharmacological inhibitors selectively targeted to various cellular pathways, we show in this study that engagement of CD44 induces lipid raft coalescence to facilitate a CD44-Src-integrin signaling axis in lipid rafts, leading to increased matrix-derived survival. Palmitoylation of the membrane-proximal cysteine residues and carboxyl-terminal linkage to the actin cytoskeleton both contribute to raft targeting of CD44. The enrichment of integrin beta1 in lipid rafts is tightly coupled to CD44 ligation-elicited lipid raft reorganization and associated with temporally delayed endocytosis. Through the interaction with the CD44 carboxyl-terminal ankyrin domain, Src is cotranslocated to lipid rafts, where it induces integrin activation via an inside-out mechanism. Collectively, this study demonstrates an important role of the dynamic raft reorganization induced by CD44 clustering in eliciting the matrix-derived survival signal.
Asunto(s)
Supervivencia Celular , Matriz Extracelular/metabolismo , Receptores de Hialuranos/metabolismo , Integrina beta1/metabolismo , Microdominios de Membrana/metabolismo , Transducción de Señal/fisiología , Familia-src Quinasas/metabolismo , Actinas/metabolismo , Apoptosis/fisiología , Adhesión Celular/fisiología , Línea Celular , Citoesqueleto/metabolismo , Humanos , Receptores de Hialuranos/genética , Integrina beta1/genética , Microdominios de Membrana/química , Osteopontina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Familia-src Quinasas/genéticaRESUMEN
Tumor formation can result from a decrease in cell death, as well as an increase in cell proliferation. In spite of the high incidence of mammary gland tumors (MGTs) in female dogs, the understanding of its etiology is still poor. Consistent with several proto-oncogenes (such as Wnt) for the mammary gland, sFRP2 is expressed in canine MGTs which is normally silent in the mammary gland. To elucidate the roles of SFRP2 in the tumorigenesis of MGTs, apoptosis regulation mediated by sFRP2 was investigated by overexpression of sFRP2 in MGT cells. DNA fragmentation and TUNEL assays showed a decreased susceptibility of the cells to UV-induced apoptosis in the context of sFRP2 overexpression. To analyze the pathways through which sFRP2 transduces anti-apoptosis signals, multiple-color immunofluorescence staining, immunoprecipitation, and immunoblotting were carried out. sFRP2 was found co-localized in the extracellular matrix of MGTs and the tyrosine phosphorylation of FAK was enhanced. Moreover, JNK was suppressed and NF-kB was activated in the cells expressing sFRP2 after UV-induced apoptosis analyzed by immunoblotting and electrophoretic mobility shift assay (EMSA). Taken together, these results suggest that sFRP2 exerts its anti-apoptotic function in mammary cancer cells through NF-kappaB activation or JNK suppression.
Asunto(s)
Apoptosis , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias Mamarias Animales/metabolismo , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Animales , Fragmentación del ADN , Perros , Femenino , Regulación Neoplásica de la Expresión Génica , Etiquetado Corte-Fin in Situ , Neoplasias Mamarias Animales/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Transducción de Señal , Transfección , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
Abnormal regulation of apoptosis and cell proliferation is thought to be involved in tumor formation. The secreted Frizzled-related protein 2 (SFRP2) was detected in primary culture of canine mammary gland tumors but not in normal mammary tissues. Thus, to elucidate the role of SFRP2 in mammary tumorigenesis, we overexpressed SFRP2 in mammary gland tumor and MCF7 cells. The results indicated that SFRP2 is secreted and incorporated into the extracellular matrix (ECM) of the tumor and normal cells. In an attempt to understand the molecular basis underlying the interaction between SFRP2 and ECM, co-immunoprecipitation and cell adhesion assays were carried out. SFRP2 was found to be associated with the fibronectin-integrin protein complex and could promote cell adhesion. DNA fragmentation and caspase 3 activity analyses showed that the susceptibility of the cells to UV-induced apoptosis decreased in the context of SFRP2 overexpression. Upon disruption of the fibronectin-integrin connection, the antiapoptosis activity of SFRP2 was decreased. Moreover, SFRP2 was found to induce tumorous transformation in normal mammary epithelial cells and to inhibit apoptosis in a modified paracrine model. Collectively, our results emphasize the relevance of SFRP2 and ECM in mammary tumorigenesis and provide further insight into the mechanism of SFRP2 action.
Asunto(s)
Apoptosis , Proteínas de la Membrana , Proteínas/metabolismo , Animales , Comunicación Autocrina , Western Blotting , Caspasa 3 , Caspasas/metabolismo , Adhesión Celular , División Celular , Línea Celular , Línea Celular Tumoral , ADN/metabolismo , Fragmentación del ADN , Perros , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Humanos , Integrina alfa5beta1/metabolismo , Integrinas/metabolismo , Neoplasias Mamarias Animales , Comunicación Paracrina , Plásmidos/metabolismo , Pruebas de Precipitina , Rayos UltravioletaRESUMEN
Canine mammary gland tumor (MGT) is the commonest tumor in female dogs and a good animal model of human breast cancer. A group of newly identified genes encoding secreted frizzled-related proteins (SFRP) have been implicated in apoptosis regulation and tumorigenesis. Canine mammary tissues from 50 spontaneous MGTs and 10 normal mammary glands (MGs) were obtained from surgically excised specimens and analyzed for expression of SFRP2, beta-catenin, and cyclin D1. By RT-PCR and in situ hybridization, SFRP2 gene was found abundantly expressed in neoplastic mammary tissues but not in normal mammary tissues, suggesting that SFRP2 may contribute as a tumor marker in canine MGTs. By immunohistochemical staining, the immunoreactivity of the SFRP2 protein was detected in more diverse areas than SFRP2 mRNA expression, including nuclei or/and cytoplasm and extracellular matrix of the tumor. In tumor masses, beta-catenin lost its tight association with the membrane and diffused into the nucleus. The expression of beta-catenin (79.4% positive) and cyclin D1 (71.4% positive) was also increased in MGTs. In the course of tumor progression, SFRP2 mRNA ( p < 0.05) and beta-catenin protein ( p < 0.01) steadily increased but not in cyclin D1. The level of SFRP2 was linearly correlated with its downstream target beta-catenin ( p < 0.05), but not correlated with cyclin D1 ( p < 0.5). As revealed in this study, the exclusive overexpression of SFRP2 in canine MGTs suggests that SFRP2 is a potential candidate gene for further investigation of mammary tumorigenesis and complex etiology of the canine model of mammary neoplasms.