RESUMEN
In skeletal tissue, loss or mutation of the gap junction protein connexin 43 (Cx43, also known as GJA1) in cells of the osteoblast lineage leads to a profound cortical bone phenotype and defective tissue remodeling. There is mounting evidence in bone cells that the C-terminus (CT) of Cx43 is a docking platform for signaling effectors and is required for efficient downstream signaling. Here, we examined this function, using a mouse model of Cx43 CT-truncation (Gja1 K258Stop). Relative to Gja1+/- controls, male Gja1-/K258Stop mice have a cortical bone phenotype that is remarkably similar to those reported for deletion of the entire Cx43 gene in osteoblasts. Furthermore, we show that the Cx43 CT binds several signaling proteins that are required for optimal osteoblast function, including PKCδ, ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) and ß-catenin. Deletion of the Cx43 CT domain affects these signaling cascades, impacting osteoblast proliferation, differentiation, and collagen processing and organization. These data imply that, at least in bone, Cx43 gap junctions not only exchange signals, but also recruit the appropriate effector molecules to the Cx43 CT in order to efficiently activate signaling cascades that affect cell function and bone acquisition.
Asunto(s)
Remodelación Ósea , Conexina 43/química , Conexina 43/metabolismo , Osteoblastos/metabolismo , Osteogénesis , Transducción de Señal , Animales , Resorción Ósea/patología , Calcificación Fisiológica , Diferenciación Celular , Proliferación Celular , Colágeno/metabolismo , Hueso Cortical/metabolismo , Matriz Extracelular/metabolismo , Masculino , Ratones , Modelos Animales , Fenotipo , Porosidad , Unión Proteica , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Osteoblasts, osteocytes, and osteoprogenitor cells are interconnected into a functional network by gap junctions formed primarily by connexin43 (Cx43). Over the past two decades, it has become clear that Cx43 is important for the function of osteoblasts and osteocytes. This connexin contributes to the acquisition of peak bone mass and is a major modulator of cortical modeling. We review key data from human and mouse genetics on the skeletal consequences of ablation or mutation of the Cx43 gene (Gja1) and the molecular mechanisms by which Cx43 regulates the differentiation, function, and survival of osteogenic lineage cells. We also discuss putative second messengers that are communicated by Cx43 gap junctions, the role of hemichannels, and the function of Cx43 as a scaffold for signaling molecules. Current knowledge demonstrates that Cx43 is more than a passive channel; rather, it actively participates in the generation and modulation of cellular signals that drive skeletal development and homeostasis.
Asunto(s)
Diferenciación Celular/fisiología , Conexina 43/metabolismo , Osteoblastos/metabolismo , Osteocitos/citología , Transducción de Señal/fisiología , Animales , Comunicación Celular/fisiología , Conexina 43/genética , Humanos , Osteoblastos/citologíaRESUMEN
The cytoplasmic C-terminus of connexin43 (Cx43) interacts with numerous signaling complexes. We hypothesize that signal complex docking to the Cx43 C-terminus (CT) is required to propagate the molecules being shared by gap junctions. We have previously shown that Cx43 impacts the responsiveness of osteoblasts to FGF2 in a PKCδ- and ERK-dependent manner, converging on Runx2 activity. Here, we mapped the interaction domain of Cx43 and PKCδ to amino acids 243-302 of the Cx43 CT by GST pulldown assay. Using Runx2-responsive luciferase reporter assays, a Cx43 deletion construct (Cx43 S244Stop), which lacks the C-terminus (amino acids 244-382), failed to support the Cx43-dependent potentiation of transcription following FGF2 treatment in MC3T3 osteoblast-like cells. Similarly, overexpression of Cx43 S244Stop could not mimic the ability of the full length Cx43 to stimulate expression of osteoblast genes. In contrast to full length Cx43, overexpression of just the Cx43 CT (amino acids 236-382) inhibited both transcription from a Runx2 reporter and signaling via PKCδ and ERK. Inhibition of signaling by the CT did not occur in HeLa cells, which lack endogenous Cx43. In summary, the data support a model in which an intact Cx43 is required for both signal propagation/permeability (i.e., channel function) and local recruitment of signaling complexes to the CT (i.e., docking function) in order to mediate its cellular effects. Further, while the CT alone has channel independent activity, it is opposing to the effect of overexpression of the full length Cx43 channel in this cell context.
Asunto(s)
Conexina 43/metabolismo , Osteoblastos/metabolismo , Animales , Western Blotting , Comunicación Celular/genética , Comunicación Celular/fisiología , Línea Celular , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Uniones Comunicantes/metabolismo , Ratones , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiologíaRESUMEN
The gap junction protein, connexin43 (Cx43), plays an important role in skeletal biology. Previously, we have shown that Cx43 can enhance the signaling and transcriptional response to fibroblast growth factor 2 (FGF2) in osteoblasts by increasing protein kinase C-δ (PKCδ) activation to affect Runx2 activity. In the present study, we show by luciferase reporter assays that the ERK signaling cascade acts in parallel to PKCδ to modulate Runx2 activity downstream of the Cx43-dependent amplification of FGF2 signaling. The PKCδ-independent activation of ERK by FGF2 was confirmed by Western blotting, as was the Cx43-dependent enhancement of ERK activation. Consistent with our prior observations for PKCδ, flow cytometry analyses show that Cx43 overexpression enhances the percentage of phospho-ERK-positive cells in response to FGF2, supporting the notion that shared signals among gap junction-coupled cells result in the enhanced response to FGF2. Western blots and luciferase reporter assays performed on osteoblasts cultured under low-density and high-density conditions revealed that cell-cell contacts are required for Cx43 to amplify ERK activation and gene transcription. Similarly, inhibition of gap junctional communication with the channel blocker 18ß-glycyrrhetinic acid attenuates the Cx43-dependent enhancement of Runx2-transcriptional activity. In total, these data underscore the importance of cell-cell communication and activation of the ERK and PKCδ pathways in the coordination of the osteoblast response to FGF2 among populations of osteoblasts.
Asunto(s)
Conexina 43/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Osteoblastos/metabolismo , Proteína Quinasa C-delta/metabolismo , Animales , Comunicación Celular/fisiología , Células Cultivadas , Conexina 43/genética , Conexinas/genética , Conexinas/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Uniones Comunicantes/genética , Uniones Comunicantes/metabolismo , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacología , Ratones , Osteoblastos/efectos de los fármacos , Proteína Quinasa C-delta/genética , Transducción de Señal , Transcripción GenéticaRESUMEN
The purpose of this study was to characterize the molecular phenotype that occurs during the profound morphological shift of cultured osteogenic cells upon treatment with fibroblast growth factor-2 (FGF2). A time course of treatment with FGF2 was performed on an osteoblast cell line, primary bone marrow stromal cells and an osteocyte-like cell line. Morphologic changes were recorded, and gene profiling was carried out by real time PCR. By 8h of FGF2 treatment, there is a striking morphological shift of osteoblast and stromal cells to an elongated dendritic-like morphology that is remindful of osteocytes. In osteoblasts treated with FGF2, this morphologic shift is preceded by an induction of several osteocyte markers, including dentin matrix protein 1 (>20-fold) and E11 (>5-fold). There is a transient increase in the gene expression of sclerostin (3.5-fold) and PHEX (2.5-fold). Sclerostin regulation by FGF2 is complex, as gene expression becomes markedly inhibited by FGF2 at times points after 8h of treatment before rebounding at day 12. Analogous modulation of osteocyte markers is seen in bone marrow stromal cells and MLO-Y4 osteocyte-like cells. In conclusion, this study shows that FGF2 can regulate the transition of osteogenic cells towards the osteocyte lineage, as well as, regulate the expression of critical genes in osteocytes.
Asunto(s)
Diferenciación Celular/genética , Factor 2 de Crecimiento de Fibroblastos/fisiología , Regulación de la Expresión Génica , Osteoblastos/fisiología , Osteocitos/citología , Proteínas Adaptadoras Transductoras de Señales , Animales , Biomarcadores , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , Proteínas Morfogenéticas Óseas/genética , Células Cultivadas , Proteínas de la Matriz Extracelular/genética , Factor 2 de Crecimiento de Fibroblastos/farmacología , Perfilación de la Expresión Génica , Marcadores Genéticos/genética , Glicoproteínas , Péptidos y Proteínas de Señalización Intercelular , Ratones , Osteoblastos/efectos de los fármacos , Endopeptidasa Neutra Reguladora de Fosfato PHEX/genéticaRESUMEN
BACKGROUND: We have recently demonstrated that modulation of the gap junction protein, connexin43, can affect the response of osteoblasts to fibroblast growth factor 2 in a protein kinase C-delta-dependent manner. Others have shown that the C-terminal tail of connexin43 serves as a docking platform for signaling complexes. It is unknown whether protein kinase C-delta can physically interact with connexin43. RESULTS: In the present study, we investigate by immunofluorescent co-detection and biochemical examination the interaction between Cx43 and protein kinase C-delta. We establish that protein kinase C-delta physically interacts with connexin43 during fibroblast growth factor 2 signaling, and that protein kinase C delta preferentially co-precipitates phosphorylated connexin43. Further, we show by pull down assay that protein kinase C-delta associates with the C-terminal tail of connexin43. CONCLUSIONS: Connexin43 can serve as a direct docking platform for the recruitment of protein kinase C-delta in order to affect fibroblast growth factor 2 signaling in osteoblasts. These data expand the list of signal molecules that assemble on the connexin43 C-terminal tail and provide a critical context to understand how gap junctions modify signal transduction cascades in order to impact cell function.
Asunto(s)
Conexina 43/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteína Quinasa C-delta/metabolismo , Transducción de Señal , Animales , Inmunoprecipitación , Osteoblastos/metabolismo , Fosforilación , Dominios y Motivos de Interacción de ProteínasRESUMEN
BACKGROUND: HMGA2 expression has been shown to be associated with enhanced selective chemosensitivity towards the topoisomerase (topo) II inhibitor, doxorubicin, in cancer cells. Although the roles of signaling cascades and proteins as regulatory factors in development, neoplasia and adaptation to the environment are becoming well established, evidence for the involvement of regulatory small RNA molecules, such as microRNAs (miRNAs) as important regulators of both transcriptional and posttranscriptional gene silencing is presently mounting. RESULTS: Here we report that HMGA2 expression in head and neck squamous cell carcinoma (HNSCC) cells is regulated in part by miRNA-98 (miR-98). Albeit HMGA2 is associated with enhanced selective chemosensitivity towards topoisomerase (topo) II inhibitor, doxorubicin in HNSCC, the expression of HMGA2 is thwarted by hypoxia. This is accompanied by enhanced expression of miRNA-98 and other miRNAs, which predictably target HMGA2. Moreover, we show that transfection of pre-miR-98trade mark during normoxia diminishes HMGA2 and potentiates resistance to doxorubicin and cisplatin. These findings implicate the role of a miRNA as a key element in modulating tumors in variable microenvironments. CONCLUSION: These studies validate the observation that HMGA2 plays a prominent role in governing genotoxic responses. However, this may only represent cells growing under normal oxygen tensions. The demonstration that miRNA profiles are altered during hypoxia and repress a genotoxic response indicates that changes in microenvironment in eukaryotes mimic those of lower species and plants, where, for example, abiotic stresses regulate the expression of thousands of genes in plants at both transcriptional and posttranscriptional levels through a number of miRNAs and other small regulatory RNAs.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Proteína HMGA2/genética , Proteína HMGA2/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , MicroARNs/metabolismo , Secuencia de Bases , Northern Blotting , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
BACKGROUND: Polymorphisms or mutations in hypoxia inducible factor-1 alpha (HIF-1alpha) that increases its activity and stability under normoxia have recently been identified. Likewise, disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 has been shown to result in abnormal accumulation of HIF-1alpha. Here, we investigate the novel polymorphisms in exon 12, that approximate the oxygen-dependent degradation domain of HIF-1alpha in five cell lines and 28 patients with oral squamous carcinomas. Moreover, we assess for the presence of polymorphisms and mutations in TSC1 and TSC2, to ascertain if dysregulation of such might complement HIF-1alpha expression. RESULTS: Denaturing high pressure liquid chromatography (DHPLC) analysis on PCR fragments in exon 12 of HIF-1alpha from 28 patients with OSCC revealed that 6 of 28 patients had mismatched heteroduplex patterns. Genomic DNA was extracted from peripheral blood leukocytes and direct sequencing showed that in 5 of the six cases these changes represented polymorphisms while, one case was a somatic mutation. Analyses of TSC1 and TSC2 revealed heteroduplexes in exons: TSC1 exon 17; TSC2 exons 36, 40, and 41. The relative levels of HIF-1alpha were significantly greater for tumors possessing a HIF-1alpha polymorphism or mutation within exon 12, whereas tumors possessing a deletion or polymorphism in TSC1/TSC2 displayed a trend for higher levels of HIF-1alpha. Western blot analyses for HIF-1alpha, TSC1 and TSC2 in five SCC cell lines revealed high levels of HIF-1alpha in SCC cells possessing TSC1 and/or TSC2 mutations. Wild-type TSC2 cells targeted with siRNA to TSC2 exhibited increased levels of HIF-1alpha. Transfection of a HIF-1alpha mutant produced higher levels of HIF-1alpha in TSC1/TSC2 mutant cell lines than in wild type cells. TSC1/TSC2 mutant cell lines administered Rapamycin blocked S6 phorphorylation and diminished the levels of HIF-1alpha to those observed in cell lines with wild type TSC1/TSC2. CONCLUSION: Dysregulation of the TSC1/TSC2 complex by mutation compliments HIF-1alpha polymorphisms in the expression of HIF-1alpha in SCC of the head and neck, and may provide biomarkers to predict responses to specific therapies and overall disease prognosis.
Asunto(s)
Carcinoma de Células Escamosas/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias de la Boca/genética , Proteínas Supresoras de Tumor/genética , Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Femenino , Neoplasias de Cabeza y Cuello , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Neoplasias de la Boca/sangre , Neoplasias de la Boca/patología , Mutación , Polimorfismo Genético , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Interferencia de ARN , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Transfección , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Human squamous cell carcinoma of the head and neck (SCCHN) is characterized by over expression of a tumor cell surface-specific receptor namely Hsp47/CBP2 that makes it a favorable candidate for targeted delivery of anticancer drugs. Several synthetic peptides have been identified as effective ligands for binding to CBP2. The purpose of this study is to investigate the potential of water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates containing a Hsp47/CBP2 binding peptide sequence, namely WHYPWFQNWAMA for targeted delivery to SCCHN. An HPMA copolymer containing Dox and CBP2 targeting peptide conjugated via lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer was synthesized by free radical precipitation copolymerization. A control polymer without targeting moiety was also synthesized. The conjugates were characterized for drug content, peptide content, molecular weight and molecular weight distribution. The uptake of polymeric conjugates by both drug resistant and drug sensitive SCCHN cells were determined in vitro by flow cytometry using FACS scan analysis. Cytotoxicity of the conjugates towards drug sensitive as well as multidrug resistant SCCHN cells were evaluated by a clonal survival assay and compared to free Dox. The cytotoxicity of the free peptide was similarly evaluated. The internalization and subcellular fate of the conjugates in drug sensitive SCCHN cells was monitored using confocal microscopy. The new targetable copolymer contained 0.16 mmole peptide/g polymer. Studies on drug sensitive SCCHN cells demonstrated lesser uptake of both targeted and non-targeted conjugates compared to free Dox suggesting a slower endocytic mechanism of uptake for the conjugates as opposed to rapid diffusion of free Dox. At higher Dox equivalent concentrations (>20 microM) the targeted conjugate showed significantly higher uptake (p < or = 0.028) than the non-targeted conjugate. The uptake of the targeted conjugate was inhibited in the presence of an anti Hsp47 antibody suggesting the involvement of active receptor mediated endocytosis in cell entry of the conjugate. Compared to free Dox, the targeted and non-targeted conjugates caused marginally lower inhibition (p < or = 0.01) of the drug sensitive SCCHN cells. In contrast, the same conjugates showed significantly higher uptake (p < or = 0.004) by drug resistant SCCHN cells and caused significantly higher inhibition (p < or = 0.02) of drug resistant SCCHN cells when compared to free Dox. Results suggest that the polymeric conjugates were able to overcome drug resistance. Confocal microscopy studies demonstrated the uptake of the polymeric conjugates, followed by internalization, intralysosomal localization and subsequent release of Dox. HPMA copolymer-Dox-peptide conjugates targeted to SCCHN cells were able to overcome drug resistance and increase efficacy in vitro. The results suggest that targetable polymeric conjugates have potential to improve systemic head and neck cancer chemotherapy by increasing tumor localization and reducing dose-limiting toxicity.
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/farmacocinética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fenómenos Químicos , Química Farmacéutica , Química Física , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Excipientes , Citometría de Flujo , Proteínas del Choque Térmico HSP47 , Proteínas de Choque Térmico/metabolismo , Humanos , Metacrilatos , Microscopía Confocal , Serpinas/metabolismo , Fracciones Subcelulares/metabolismo , Ensayo de Tumor de Célula MadreRESUMEN
The mechanical environment is known to influence fracture healing. We speculated that connexin43 (Cx43) gap junctions, which impact skeletal homeostasis, fracture healing and the osteogenic response to mechanical load, may play a role in mediating the response of the healing bone to mechanical strain. Here, we used an established rat fracture model, which uses a 2 mm osteotomy gap stabilized by an external fixator, to examine the impact of various cyclical axial loading protocols (2%, 10%, and 30% strain) on osteotomy healing. We examined the presence of Cx43 in the osteotomy-healing environment and assessed how mechanical strain modulates Cx43 expression patterns in the callus. We demonstrated that increased cyclical axial strain results in increased radiographic and histologic bone formation. In addition, we show by immunohistochemistry that Cx43 is abundantly expressed in the healing callus, with the expression most robust in samples exposed to increased cyclical axial strain. These data are consistent with the concept that an increase in Cx43 expression by mechanical load may be part of the mechanisms by which mechanical forces enhances fracture healing.
RESUMEN
Connexin43 (Cx43) plays a critical role in osteoblast function and bone mass accrual, yet the identity of the second messengers communicated by Cx43 gap junctions, the targets of these second messengers and how they regulate osteoblast function remain largely unknown. We have shown that alterations of Cx43 expression in osteoblasts can impact the responsiveness to fibroblast growth factor-2 (FGF2), by modulating the transcriptional activity of runt-related transcription factor 2 (Runx2). In this study, we examined the contribution of the phospholipase Cγ1/inositol polyphosphate/protein kinase C delta (PKCδ) cascade to the Cx43-dependent transcriptional response of MC3T3 osteoblasts to FGF2. Knockdown of expression and/or inhibition of function of phospholipase Cγ1, inositol polyphosphate multikinase, which generates inositol 1,3,4,5-tetrakisphosphate (InsP4) and InsP5, and inositol hexakisphosphate kinase 1/2, which generates inositol pyrophosphates, prevented the ability of Cx43 to potentiate FGF2-induced signaling through Runx2. Conversely, overexpression of phospholipase Cγ1 and inositol hexakisphosphate kinase 1/2 enhanced FGF2 activation of Runx2 and the effect of Cx43 overexpression on this response. Disruption of these pathways blocked the nuclear accumulation of PKCδ and the FGF2-dependent interaction of PKCδ and Runx2, reducing Runx2 transcriptional activity. These data reveal that FGF2-signaling involves the inositol polyphosphate cascade, including inositol hexakisphosphate kinase (IP6K), and demonstrate that IP6K regulates Runx2 and osteoblast gene expression. Additionally, these data implicate the water-soluble inositol polyphosphates as mediators of the Cx43-dependent amplification of the osteoblast response to FGF2, and suggest that these low molecular weight second messengers may be biologically relevant mediators of osteoblast function that are communicated by Cx43-gap junctions.
Asunto(s)
Conexina 43/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Osteoblastos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Proteína Quinasa C-delta/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular , Conexina 43/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica/fisiología , Humanos , Fosfatos de Inositol/genética , Fosfatos de Inositol/metabolismo , Ratones , Osteoblastos/citología , Fosfolipasa C gamma/genética , Fosfolipasa C gamma/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Proteína Quinasa C-delta/genéticaRESUMEN
The transcription factor osterix (Osx/Sp7) is required for osteogenic differentiation and bone formation in vivo. While Osx can act at canonical Sp1 DNA-binding sites and/or interact with NFATc1 to cooperatively regulate transcription in some osteoblast promoters, little is known about the molecular details by which Osx regulates osteocalcin (OCN) transcription. We previously identified in the OCN proximal promoter a minimal C/T-rich motif, termed OCN-CxRE (connexin-response element) that binds Sp1 and Sp3 in a gap junction-dependent manner. In the present study, we hypothesized that Osx could act via this non-canonical Sp1/Sp3-binding element to regulate OCN transcription. OCN promoter luciferase reporter assays show that Osx alone is an insufficient activator that requires Sp1, but not Sp3, to synergistically stimulate OCN promoter activity. Moreover, promoter deletion analyses demonstrate that both the Sp1/Sp3-binding OCN-CxRE (-70 to -57) and the -92 to -87 region of the OCN proximal promoter are critical for Osx/Sp1 synergistic activities. Our data show that Sp1 influences Osx activity by enhancing Osx occupancy on the OCN promoter, perhaps via physical interactions between the two transcription factors. Finally, alteration of the expression of the gap junction protein connexin43 modulates the recruitment of both Sp1 and Osx to the OCN promoter. In total, our data are strongly in support of Sp1 as an essential transcription factor required for Osx recruitment and transactivation of the OCN promoter. Further, these data lend insight into a mechanism by which alteration of connexin43 impacts osteogenesis in vitro and in vivo.
Asunto(s)
Osteocalcina/genética , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Conexina 43/metabolismo , ADN/metabolismo , Uniones Comunicantes/metabolismo , Ratones , Datos de Secuencia Molecular , Unión Proteica , Ratas , Factor de Transcripción Sp3/metabolismo , Factor de Transcripción Sp7RESUMEN
In this study, we examine the role of the gap junction protein, connexin43 (Cx43), in the transcriptional response of osteocalcin to fibroblast growth factor 2 (FGF2) in MC3T3 osteoblasts. By luciferase reporter assays, we identify that the osteocalcin transcriptional response to FGF2 is markedly increased by overexpression of Cx43, an effect that is mediated by Runx2 via its OSE2 cognate element, but not by a previously identified connexin-responsive Sp1/Sp3-binding element. Furthermore, disruption of Cx43 function with Cx43 siRNAs or overexpression of connexin45 markedly attenuates the response to FGF2. Inhibition of protein kinase C delta (PKCdelta) with rottlerin or siRNA-mediated knockdown abrogates the osteocalcin response to FGF2. Additionally, we show that upon treatment with FGF2, PKCdelta translocates to the nucleus, PKCdelta and Runx2 are phosphorylated and these events are enhanced by Cx43 overexpression, suggesting that the degree of activation is enhanced by increased Cx43 levels. Indeed, chromatin immunoprecipitations of the osteocalcin proximal promoter with antibodies against Runx2 demonstrate that the recruitment of Runx2 to the osteocalcin promoter in response to FGF2 treatment is dramatically enhanced by Cx43 overexpression. Thus, Cx43 plays a critical role in regulating the ability of osteoblasts to respond to FGF2 by impacting PKCdelta and Runx2 function.
Asunto(s)
Conexina 43/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Osteoblastos/efectos de los fármacos , Proteína Quinasa C-delta/metabolismo , Acetofenonas/farmacología , Animales , Benzopiranos/farmacología , Línea Celular , Inmunoprecipitación de Cromatina , Conexina 43/genética , Conexinas/genética , Conexinas/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Inhibidores Enzimáticos/farmacología , Immunoblotting , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Microscopía Fluorescente , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/genética , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteína Quinasa C-delta/genética , ARN Interferente Pequeño/genética , Ratas , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
Low pO(2) values are a common finding among oral squamous cell carcinomas (SCC). Our objective was to determine the role that oxygen tension plays on the direct tumor effect of endostatin (ES). Squamous carcinoma cell lines were grown under normoxic or hypoxic conditions and treated with endostatin (ES), nitric oxide (NO) donors, NO scavengers, NO synthase inhibitors, or transduced with AdenoVec-hEndo or AdenoVec Null vectors. The expression of vascular endothelial growth factor (VEGF) and collagen XVIII were determined by RT-PCR and protein levels assessed by Western blot analyses. Our studies demonstrated that collagen XVIII and VEGF are expressed and responsive to ES in a limited number of SCC cell lines during normoxia but were most responsive when grown under hypoxic conditions. VEGF and collagen XVIII were downregulated by both ES and transduction of cells with AdenoVec-hEndo. The effects of ES on SCC cells were enhanced by aminoguanidine (Ag), L-NAME, and diphenyleneiodonium chloride (DPI). Endostatin and transduced with ES vectors diminished the levels of NO whereas NO donors enhanced VEGF expression and collagen XVIII expression. In conclusion, the direct effect of endostatins on tumor cells is most effective under conditions of low oxygen tension and can be potentiated by the use of nitric oxide synthase inhibitors or NO scavengers.
Asunto(s)
Colágeno Tipo XVIII/genética , Endostatinas/farmacología , Regulación Neoplásica de la Expresión Génica , Óxido Nítrico/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Secuencia de Bases , Carcinoma de Células Escamosas , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Cartilla de ADN , Humanos , Invasividad Neoplásica/prevención & control , Donantes de Óxido Nítrico/farmacología , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
CBP2/Hsp47 is a glycoprotein normally limited to the ER-Golgi where it is first associated with procollagen chains at a very early point during translation of nascent chains and later with properly folded procollagen. Although CBP2/Hsp47 is regarded as a molecular chaperone belonging to the serpin superfamily, this protein does not appear to inhibit serine proteinases. Here we demonstrate that CBP2/Hsp47 functions in a manner similar to other serpin superfamily members by cross class inhibiting cysteine proteinases. A CBP2/Hsp47 to cathepsin L inactivation stoichiometery of approximately 1.5 revealed concurrent cleavage of CBP2/Hsp47 with proteinase inactivation. Cleavage of the CBP2/Hsp47 was shown to occur outside the P1-P1' at the P16-P15 and P2'-P3' bonds. In addition, the proteinase bands in SDS/PAGE diminished on reaction of the enzyme with CBP2/Hsp47. These results sustain a mechanism advocated by Bjork et al. (1998), in which cysteine proteinases assault a peptide bond in the reactive site loop of serpins, (CBP2/Hsp47) adjacent to the P1-P1' bonds involved in serine proteinase inhibition. The reaction proceeds with the substrate pathway dominating in the cysteine proteinase reaction. In these complexes the cysteine proteinases, papain and cathepsin L, are rendered more susceptible to proteolysis and are degraded by active enzyme. These properties help explain the mechanism by which CBP2/Hsp47 increases the fidelity of collagen production. Moreover, if CBP2/Hsp47 is shown to involve the multiplexin subclass of collagens, it may further provide a mechanism by which the motogen and angiogenic properties during development and/or neoplasia are regulated.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Proteínas de Choque Térmico/metabolismo , Carcinoma de Células Escamosas/metabolismo , Catepsina L , Catepsinas/antagonistas & inhibidores , Electroforesis en Gel de Poliacrilamida , Proteínas del Choque Térmico HSP47 , Humanos , Células Tumorales Cultivadas/metabolismoRESUMEN
There is strong evidence that nonsteroidal antiinflammatory drug (NSAID) sulindac may exert a significant antineoplastic effect. The purpose of our study was to explore the effects of sulindac on human oral squamous cell carcinoma (SCCa) cells and to elucidate the underlying molecular mechanisms. The changes that sulindac treatment induced on growth, apoptosis and cell cycle distribution of human oral SCCa cell lines were assessed by cell growth and flow cytometry experiments. Utilizing quantitative RT-PCR and immunocytochemistry, we determined the effect of sulindac treatment on mRNA and protein expression of different sulindac's targets. Also, PPARgamma expression was selectively targeted by antisense oligonucleotide treatment. Both sulfide and sulfone metabolites of sulindac, which differ in the ability to cause COX-2 inhibition, induced a significant dose- and time-dependent cell growth reduction accompanied by increase in apoptosis without concomitant cell cycle arrest. Sulindac treatment also caused upregulation of the protein and mRNA expression levels of COX-2 and PPARs. Treatment with antisense PPARgamma oligonucleotides abolished sulindac's growth inhibitory effect. Our results are consistent with a significant growth inhibitory effect of NSAID sulindac on human oral SCCa cells, which is mediated, at least partially, through induction of apoptosis. We suggest that upregulation of PPARgamma expression and activation may be, at least partially, responsible for sulindac's antiproliferative effect.