RESUMO
PHYTOCHROME INTERACTING FACTORs (PIFs) are a group of basic helix-loop-helix (bHLH) transcription factors that repress plant light responses. PIF8 is one of the less-characterized Arabidopsis (Arabidopsis thaliana) PIFs, whose putative orthologs are conserved in other plant species. PIF8 possesses a bHLH motif and an active phytochrome B motif but not an active phytochrome A motif. Consistent with this motif composition, PIF8 binds to G-box elements and interacts with the Pfr form of phyB but only very weakly, if at all, with that of phyA. PIF8 differs, however, from other PIFs in its protein accumulation pattern and functional roles in different light conditions. First, PIF8 inhibits phyA-induced seed germination, suppression of hypocotyl elongation, and randomization of hypocotyl growth orientation in far-red light, but it does not inhibit phyB-induced red light responses. Second, PIF8 protein accumulates more in far-red light than in darkness or red light. This is distinct from the pattern observed with PIF3, which accumulates more in darkness. This PIF8 accumulation pattern requires degradation of PIF8 by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in darkness, inhibition of COP1 by phyA in far-red light, and promotion of PIF8 degradation by phyB in red light. Together, our results indicate that PIF8 is a genuine PIF that represses phyA-mediated light responses.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Luz , Fitocromo A/metabolismo , Fitocromo/metabolismo , Escuridão , Regulação da Expressão Gênica de Plantas , Hipocótilo , Fitocromo B , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Seedling hypocotyls display negative gravitropism in the dark but agravitropism in the light. The Arabidopsis thaliana pif quadruple mutant (pifQ), which lacks four PHYTOCHROME-INTERACTING FACTORS (PIFs), is agravitropic in the dark. Endodermis-specific expression of PIF1 rescues gravitropism in pifQ mutant seedlings. Since phytochromes induce light responses by inhibiting PIFs and the COP1-SPA ubiquitin E3 ligase complex in the nucleus, we asked whether phyB can cell autonomously inhibit hypocotyl negative gravitropism in the endodermis. We found that while epidermis-specific expression of PHYB rescues hypocotyl negative gravitropism and all other phyB mutant phenotypes, endodermis-specific expression of PHYB does not. Epidermal phyB induces the phosphorylation and degradation of endodermal PIFs in response to red light. This induces a global gene expression pattern similar to that induced by red light treatment of seedlings expressing PHYB under the control of its own endogenous promoter. Our results imply that epidermal phyB generates an unidentified mobile signal that travels to the endodermis where it promotes PIF degradation and inhibits hypocotyl negative gravitropism.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Hipocótilo/metabolismo , Fitocromo B/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Gravitropismo/genética , Gravitropismo/fisiologia , Hipocótilo/genética , Fitocromo B/genéticaRESUMO
The emerging roles of integrin ß3 in the epithelial-mesenchymal transition (EMT) and drug resistance underline its significance in cancer metastasis and recurrence. However, the molecular mechanism underlying the distinctive expression of integrin ß3 is less understood. In the present report, we demonstrated that repetitive exposure to transforming growth factor ß (TGFß), a potent inducer of the EMT, significantly increased the expression of integrin ß3 in A549 lung cancer cells with distinct mesenchymal properties, such as actin filament reorganization and invasiveness. Notably, integrin ß3 expression was associated to cancer cell invasion and migration, and was determined not by Smad4-dependent pathways but by sustained ERK1/2 activity in the mesenchymal cancer cells. These data suggest that ERK1/2 plays an important role in mediating non-canonical TGFß signal pathways for integrin ß3 expression. Therefore, the targeting of the MEK/ERK activity seems to be a promising therapeutic approach to suppressing EMT-associated cancer progression that potentially occurs in TGFß-enriched microenvironments, which would lead to the suppression of the metastatic potential of integrin ß3-positive cancer cells.
Assuntos
Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Integrina beta3/metabolismo , Neoplasias Pulmonares/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Integrina beta3/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Invasividade Neoplásica , Fenótipo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Microambiente Tumoral , Regulação para CimaRESUMO
Chronic exposure to TGFß, a frequent occurrence for tumor cells in the tumor microenvironment, confers more aggressive phenotypes on cancer cells by promoting their invasion and migration while at the same time increasing their resistance to the growth-inhibitory effect of TGFß. In this study, a transdifferentiated (TD) A549 cell model, established by chronically exposing A549 cells to TGFß, showed highly invasive phenotypes in conjunction with attenuation of Smad-dependent signaling. We show that Snail protein, the mRNA expression of which strongly correlates with a poor prognosis in lung cancer patients, was highly stable in TD cells after TGFß stimulation. The increased protein stability of Snail in TD cells correlated with elevated inhibitory phosphorylation of GSK3ß, resulting from the high Akt activity. Notably, integrin ß3, whose expression was markedly increased upon sustained exposure to TGFß, was responsible for the high Akt activity as well as the increased Snail protein stability in TD cells. Consistently, clinical database analysis on lung cancer patients revealed a negative correlation between overall survival and integrin ß3 mRNA levels. Therefore, we suggest that the integrin ß3-Akt-GSK3ß signaling axis plays an important role in non-canonical TGFß signaling, determining the invasive properties of tumor cells chronically exposed to TGFß.
Assuntos
Adenocarcinoma/patologia , Neoplasias Pulmonares/patologia , Transdução de Sinais/fisiologia , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células A549 , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Integrina beta3/metabolismo , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Invasividade Neoplásica/patologia , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
While metastasis, the main cause of lung cancer-related death, has been extensively studied, the underlying molecular mechanism remains unclear. A previous clinicogenomic study revealed that expression of N-acetylgalactosaminyltransferase (GalNAc-T14), is highly inversely correlated with recurrence-free survival in those with non-small cell lung cancer (NSCLC). However, the underlying molecular mechanism(s) has not been determined. Here, we showed that GalNAc-T14 expression was positively associated with the invasive phenotype. Microarray and biochemical analyses revealed that HOXB9, the expression of which was increased in a GalNAc-T14-dependent manner, played an important role in metastasis. GalNAc-T14 increased the sensitivity of the WNT response and increased the stability of the ß-catenin protein, leading to induced expression of HOXB9 and acquisition of an invasive phenotype. Pharmacological inhibition of ß-catenin in GalNAc-T14-expressing cancer cells suppressed HOXB9 expression and invasion. A meta-analysis of clinical genomics data revealed that expression of GalNAc-T14 or HOXB9 was strongly correlated with reduced recurrence-free survival and increased hazard risk, suggesting that targeting ß-catenin within the GalNAc-T14/WNT/HOXB9 axis may be a novel therapeutic approach to inhibit metastasis in NSCLC.
Assuntos
Adenocarcinoma/enzimologia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Movimento Celular , Proteínas de Homeodomínio/metabolismo , Neoplasias Pulmonares/enzimologia , N-Acetilgalactosaminiltransferases/metabolismo , Via de Sinalização Wnt , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Adenocarcinoma de Pulmão , Animais , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Bases de Dados Genéticas , Intervalo Livre de Doença , Perfilação da Expressão Gênica/métodos , Xenoenxertos , Proteínas de Homeodomínio/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , N-Acetilgalactosaminiltransferases/genética , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Estabilidade Proteica , Pirimidinonas/farmacologia , Fatores de Tempo , Transfecção , Resultado do Tratamento , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidores , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Loss of E-cadherin, a hallmark of epithelial-mesenchymal transition (EMT), can significantly affect metastatic dissemination. However, the molecular mechanism of EMT-associated metastatic dissemination by loss of E-cadherin still remains unclear in non-small cell lung cancers (NSCLCs). In the present study, we show that the knockdown of E-cadherin was sufficient to convert A549 NSCLC cells into mesenchymal type with the concurrent up-regulation of typical EMT inducers such as ZEB1 and TWIST1. Interestingly, the EMT-induced cells by E-cadherin depletion facilitate invasion in a matrix metalloproteinase-2 (MMP2)-dependent manner with aberrant activation of EGFR signaling. We demonstrated that the elevated invasiveness was a result of the activated EGFR-MEK/ERK signaling, which in turn leads to ZEB1 dependent MMP2 induction. These results suggest that the EGFR-MEK/ERK/ZEB1/MMP2 axis is responsible for promoted invasion in EMT-induced NSCLCs. Consistently, ERK activation and loss of E-cadherin were both observed in the disseminating cancer cells at the invasive tumor fronts in NSCLS cancer tissues. Thereby, these data suggest that the EGFR-MEK/ERK signaling would be a promising molecular target to control aberrant MMP2 expression and consequent invasion in the EMT-induced NSCSLs.
Assuntos
Caderinas/deficiência , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Metaloproteinase 2 da Matriz/metabolismo , Caderinas/genética , Caderinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/genéticaRESUMO
The metastatic potential of non-small cell lung cancer (NSCLC) cells has been shown to be associated with the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment, regulating tumor cell function by secreting growth factors, chemokines, and extracellular matrix (ECM). In this study, we examined the role of CAFs in the tumor progression of NSCLC. Firstly, we established primary cultures of CAFs and matched normal fibroblasts (NFs) from patients with resected NSCLC. CAFs exhibited greater expression of the pan-mesenchymal marker α-smooth muscle actin (α-SMA) than did NFs, although they displayed similar morphology. Furthermore, we employed a direct co-culture assay with human NSCLC A549 and H358 cells, and found that CAFs were more potent in inducing the epithelial-to-mesenchymal transition (EMT) phenotype than NFs, as indicated by an elongated and disseminated appearance. CAF-induced EMT led to an increase in motility and a decrease in proliferation of NSCLC cells through SMAD family number-3 (SMAD3)-dependent up-regulation of the growth inhibitory gene p21(CIP1) [cyclin-dependent kinase inhibitor-1A (CDKN1A)] and α-SMA. Taken together, these findings provide evidence that lung CAFs have tumor-promoting capacity distinct from NFs and might play a significant role in the metastatic potential of NSCLC.
Assuntos
Adenocarcinoma Bronquioloalveolar/patologia , Carcinoma de Células Grandes/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , Transição Epitelial-Mesenquimal , Fibroblastos/patologia , Neoplasias Pulmonares/patologia , Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma Bronquioloalveolar/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Adesão Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia , Pele/metabolismo , CicatrizaçãoRESUMO
Keloids are characterised by the excessive accumulation of extracellular matrix (ECM), especially overabundant collagen formation. In keloid fibroblasts (KFs), transforming growth factor (TGF)-ß-dependent signalling is closely associated with a variety of keloid pathologic responses such as ECM production and fibroblast overgrowth. Thus, inhibition of TGF-ß signalling would be a potential therapeutic approach to prevent keloid scar formation. Thereby, we aimed to identify a novel TGF-ß signalling blocker among natural products using a simplified screening approach. We discovered that the extract of Aneilema keisak (A.K-Ex) lowered TGF-ß-dependent signalling by reducing Smad2 protein levels. Given that KFs showed altered dependency on TGF-ß for survival and proliferation, A.K-Ex-mediated reduction in Smad2 protein levels significantly inhibited the major characteristics of KFs such as cell growth, migration and collagen synthesis, suggesting that A.K-Ex exhibits possible therapeutic activity on keloids.
Assuntos
Commelinaceae , Fibroblastos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/biossíntese , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Queloide/tratamento farmacológicoRESUMO
Adult stem cells, which are characterized by self-renewal and multi-potency, are classified as specialized cell types, responsible for the regeneration of damaged tissues. There is growing evidence that senescence of stem cells (or stem cell aging) is closely associated with a variety of aging-related diseases such as tissue atrophy, degenerative diseases and onset of cancers. Alterations in the systemic environment during aging may trigger stress signaling in stem cells and reduce stem cell characteristics, resulting in loss of differentiation potential and defective self-renewal (referred to as mal-differentiation). Thus, it has been suggested that aging-related disorders such as retarded regeneration of damaged tissue and onset of cancer may result from the mal-differentiation of stem cells. In particular, many types of cancers such as leukemia, intestinal cancer, skin cancer and sarcoma have been shown to originate from adult stem cells after a variety of oncogenic challenges. This review summarizes recent studies on cancers originating from stem cells, demonstrating possible molecular mechanisms that govern the susceptibility of stem cells to oncogenic challenges.
Assuntos
Células-Tronco Adultas/fisiologia , Transformação Celular Neoplásica/metabolismo , Senescência Celular/fisiologia , Neoplasias/fisiopatologia , Envelhecimento/fisiologia , Animais , Humanos , Modelos Biológicos , Células-Tronco Neoplásicas/fisiologiaRESUMO
PIF3 is a phytochrome-interacting basic helix-loop-helix transcription factor that negatively regulates light responses, including hypocotyl elongation, cotyledon opening, and hypocotyl negative gravitropism. However, the role of PIF3 in chlorophyll biosynthesis has not been clearly defined. Here, we show that PIF3 also negatively regulates chlorophyll biosynthesis by repressing biosynthetic genes in the dark. Consistent with the gene expression patterns, the etiolated pif3 mutant accumulated a higher amount of protochlorophyllide and was bleached severely when transferred into light. The photobleaching phenotype of pif3 could be suppressed by the gun5 mutation and mimicked by overexpression of GUN5. When 4 negative phytochrome-interacting protein genes (PIF1, PIF3, PIF4, and PIF5) were mutated, the resulting quadruple mutant seedlings displayed constitutive photomorphogenic phenotypes, including short hypocotyls, open cotyledons, and disrupted hypocotyl gravitropism in the dark. Microarray analysis further confirmed that the dark-grown quadruple mutant has a gene expression pattern similar to that of red light-grown WT. Together, our data indicate that 4 phytochrome-interacting proteins are required for skotomorphogenesis and phytochromes activate photomorphogenesis by inhibiting these factors.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Clorofila/biossíntese , Fotossíntese , Fitocromo/metabolismo , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Clorofila/genética , Escuridão , Perfilação da Expressão Gênica , Morfogênese/genética , Mutação , Fotodegradação , Fotossíntese/genética , Plântula/anatomia & histologia , Plântula/genética , Plântula/crescimento & desenvolvimentoRESUMO
Phytochromes are red/far-red light photoreceptors that convert the information contained in external light into biological signals. The decoding process starts with the perception of red light, which occurs through photoisomerization of a chromophore located within the phytochrome, leading to structural changes that include the disruption of intramolecular interactions between the N- and C-terminal domains of the phytochrome. This disruption exposes surfaces required for interactions with other proteins. In contrast, the perception of far-red light reverses the photoisomerization, restores the intramolecular interaction, and closes the interacting surfaces. Light information represented by the concentration of opened interacting surfaces is converted into biological signals through the modulating activity of interacting proteins. This review summarizes plant phytochromes, phytochrome-interacting proteins, and signal transmission from phytochromes to their interacting proteins.
Assuntos
Luz , Fitocromo/metabolismo , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/metabolismo , Plantas/genética , Sementes/fisiologia , Núcleo Celular/fisiologia , Germinação , Família Multigênica , Fitocromo/genética , Transdução de SinaisRESUMO
Angiosperm seeds integrate various environmental signals, such as water availability and light conditions, to make a proper decision to germinate. Once the optimal conditions are sensed, gibberellin (GA) is synthesized, triggering germination. Among environmental signals, light conditions are perceived by phytochromes. However, it is not well understood how phytochromes regulate GA biosynthesis. Here we investigated whether phytochromes regulate GA biosynthesis through PIL5, a phytochrome-interacting bHLH protein, in Arabidopsis. We found that pil5 seed germination was inhibited by paclobutrazol, the ga1 mutation was epistatic to the pil5 mutation, and the inhibitory effect of PIL5 overexpression on seed germination could be rescued by exogenous GA, collectively indicating that PIL5 regulates seed germination negatively through GA. Expression analysis revealed that PIL5 repressed the expression of GA biosynthetic genes (GA3ox1 and GA3ox2), and activated the expression of a GA catabolic gene (GA2ox) in both PHYA- and PHYB-dependent germination assays. Consistent with these gene-expression patterns, the amount of bioactive GA was higher in the pil5 mutant and lower in the PIL5 overexpression line. Lastly, we showed that red and far-red light signals trigger PIL5 protein degradation through the 26S proteasome, thus releasing the inhibition of bioactive GA biosynthesis by PIL5. Taken together, our data indicate that phytochromes promote seed germination by degrading PIL5, which leads to increased GA biosynthesis and decreased GA degradation.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Germinação/fisiologia , Giberelinas/fisiologia , Sementes/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/efeitos da radiação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos da radiação , Regulação da Expressão Gênica de Plantas , Germinação/efeitos da radiação , Giberelinas/biossíntese , Giberelinas/metabolismo , Luz , Fitocromo/fisiologia , Sementes/efeitos da radiaçãoRESUMO
The Oct-4 gene encodes a transcription factor that is expressed in embryonic stem (ES) cells and germ cells. Oct-4 is known to function as a transcriptional activator of genes involved in maintaining an undifferentiated totipotent state and possibly in preventing expression of genes activated during differentiation. In addition, it is a putative proto-oncogene and a critical player in the genesis of human testicular germ cell tumors. Although much effort has gone toward characterizing Oct-4, there is still little known about the molecular mechanisms and the proteins that regulate Oct-4 function. To identify cofactors that control Oct-4 function in vivo, we used a recently developed bacterial two-hybrid screening system and isolated a novel ES cell-derived cDNA encoding Ewing's sarcoma protein (EWS). EWS is a proto-oncogene and putative RNA-binding protein involved in human cancers. By using glutathione-S-transferase (GST) pull-down assays, we were able to confirm the interaction between Oct-4 and EWS in vitro, and moreover, coimmunoprecipitation and colocalization studies have shown that these proteins also associate in vivo. We have mapped the EWS-interacting region to the POU domain of Oct-4. In addition, three independent sites on EWS are involved in binding to Oct-4. In this study, we report that Oct-4 and EWS are coexpressed in the pluripotent mouse and human ES cells. Consistent with its ability to bind to and colocalize with Oct-4, ectopic expression of EWS enhances the transactivation ability of Oct-4. Moreover, a chimeric protein generated by fusion of EWS (1-295) to the GAL4 DNA-binding domain significantly increases promoter activity of a reporter containing GAL4 DNA-binding sites, suggesting the presence of a strong activation domain within EWS. Taken together, our results suggest that Oct-4-mediated transactivation is stimulated by EWS.