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1.
Biochem J ; 477(17): 3131-3145, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32766732

RESUMO

The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Rabdomiossarcoma/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismo , Linhagem Celular Tumoral , Proteínas Hedgehog/genética , Humanos , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Multimerização Proteica , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Proteína GLI1 em Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/genética
2.
J Investig Dermatol Symp Proc ; 19(2): S87-S88, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30471761

RESUMO

Hedgehog (HH) signaling plays an important role both during embryonic development and adult life. It is involved in the regulation of cell differentiation, cell proliferation and tissue polarity, as well as in the maintenance of stem cells, tissue repair, and regeneration (Briscoe and Therond, 2013; Jiang and Hui, 2008). Three ligands, Indian, Sonic, and Desert HH, can activate this pathway. Binding of HH ligands to their receptor, PTCH1 (Figure 1) lift its inhibition on SMO, resulting in activation and nuclear translocation of GLI transcription factors (Javelaud et al., 2012). The vertebrate GLI gene family is composed of three distinct genes GLI1, GLI2, and GLI3, encoding Krüppel-like transcription factors. GLI proteins exhibit distinct regulations, biochemical properties, and target genes. GLI3 acts as the main repressor of the pathway in the absence of HH ligands, whereas, in their presence, GLI2 is the main HH effector that drives the expression of GLI1 (Briscoe and Therond, 2013).

3.
Life Sci Alliance ; 7(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38834194

RESUMO

Vinculin is a cytoskeletal linker strengthening cell adhesion. The Shigella IpaA invasion effector binds to vinculin to promote vinculin supra-activation associated with head-domain-mediated oligomerization. Our study investigates the impact of mutations of vinculin D1D2 subdomains' residues predicted to interact with IpaA VBS3. These mutations affected the rate of D1D2 trimer formation with distinct effects on monomer disappearance, consistent with structural modeling of a closed and open D1D2 conformer induced by IpaA. Notably, mutations targeting the closed D1D2 conformer significantly reduced Shigella invasion of host cells as opposed to mutations targeting the open D1D2 conformer and later stages of vinculin head-domain oligomerization. In contrast, all mutations affected the formation of focal adhesions (FAs), supporting the involvement of vinculin supra-activation in this process. Our findings suggest that IpaA-induced vinculin supra-activation primarily reinforces matrix adhesion in infected cells, rather than promoting bacterial invasion. Consistently, shear stress studies pointed to a key role for IpaA-induced vinculin supra-activation in accelerating and strengthening cell-matrix adhesion.


Assuntos
Adesão Celular , Adesões Focais , Vinculina , Vinculina/metabolismo , Vinculina/genética , Humanos , Adesões Focais/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Mutação , Interações Hospedeiro-Patógeno , Células HeLa , Ligação Proteica , Shigella/metabolismo , Shigella/genética , Antígenos de Bactérias/metabolismo , Antígenos de Bactérias/genética , Disenteria Bacilar/microbiologia , Disenteria Bacilar/metabolismo
4.
J Biol Chem ; 287(22): 17996-8004, 2012 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-22496449

RESUMO

The melanocyte-specific transcription factor M-MITF is involved in numerous aspects of melanoblast lineage biology including pigmentation, survival, and migration. It plays complex roles at all stages of melanoma progression and metastasis. We established previously that GLI2, a Kruppel-like transcription factor that acts downstream of Hedgehog signaling, is a direct transcriptional target of the TGF-ß/SMAD pathway and contributes to melanoma progression, exerting antagonistic activities against M-MITF to control melanoma cell invasiveness. Herein, we dissected the molecular mechanisms underlying both TGF-ß and GLI2-driven M-MITF gene repression. Using transient cell transfection experiments with M-MITF promoter constructs, chromatin immunoprecipitation, site-directed mutagenesis, and electrophoretic mobility shift assays, we identified a GLI2 binding site within the -334/-296 region of the M-MITF promoter, critical for GLI2-driven transcriptional repression. This region is, however, not needed for inhibition of M-MITF promoter activity by TGF-ß. We determined that TGF-ß rapidly repressed protein kinase A activity, thus reducing both phospho-cAMP-response element-binding protein (CREB) levels and CREB-dependent transcription of the M-MITF promoter. Increased GLI2 binding to its cognate cis-element, associated with reduced CREB-dependent transcription, allowed maximal inhibition of the M-MITF promoter via two distinct mechanisms.


Assuntos
Fatores de Transcrição Kruppel-Like/fisiologia , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/genética , Proteínas Nucleares/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Sequência de Bases , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Primers do DNA , DNA de Neoplasias/genética , Progressão da Doença , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica , Humanos , Fator de Transcrição Associado à Microftalmia/fisiologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Proteína Gli2 com Dedos de Zinco
5.
Commun Biol ; 5(1): 1068, 2022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36207615

RESUMO

TGF-ß signaling is involved in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis, representing one of the four major pathways genetically altered in 100% of PDAC cases. TGF-ß exerts complex and pleiotropic effects in cancers, notably via the activation of SMAD pathways, predominantly SMAD2/3/4. Though SMAD2 and 3 are rarely mutated in cancers, SMAD4 is lost in about 50% of PDAC, and the role of SMAD2/3 in a SMAD4-null context remains understudied. We herein provide evidence of a SMAD2/3 oncogenic effect in response to TGF-ß1 in SMAD4-null human PDAC cancer cells. We report that inactivation of SMAD2/3 in SMAD4-negative PDAC cells compromises TGF-ß-driven collective migration mediated by FAK and Rho/Rac signaling. Moreover, RNA-sequencing analyses highlight a TGF-ß gene signature related to aggressiveness mediated by SMAD2/3 in the absence of SMAD4. Using a PDAC patient cohort, we reveal that SMAD4-negative tumors with high levels of phospho-SMAD2 are more aggressive and have a poorer prognosis. Thus, loss of SMAD4 tumor suppressive activity in PDAC leads to an oncogenic gain-of-function of SMAD2/3, and to the onset of associated deleterious effects.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteína Smad3/metabolismo , Carcinogênese/genética , Carcinoma Ductal Pancreático/metabolismo , Humanos , Neoplasias Pancreáticas/metabolismo , RNA , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Neoplasias Pancreáticas
6.
J Biol Chem ; 285(1): 409-21, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19858184

RESUMO

cAMP is a key messenger of many hormones and neuropeptides, some of which modulate the composition of extracellular matrix. Treatment of human dermal fibroblasts with dibutyryl cyclic AMP and forskolin antagonized the inductive effects of transforming growth factor-beta (TGF-beta) on the expression of collagen, connective tissue growth factor, tissue inhibitor of matrix metalloproteinase-1, and plasminogen activator inhibitor type I, four prototypical TGF-beta-responsive genes. Increased intracellular cAMP prevented TGF-beta-induced Smad-specific gene transactivation, although TGF-beta-mediated Smad phosphorylation and nuclear translocation remained unaffected. However, increased cAMP levels abolished TGF-beta-induced interaction of Smad3 with its transcriptional co-activator cAMP-response element-binding protein (CREB)-binding protein (CBP)/p300. Overexpression of the transcriptional co-activator CBP/p300 rescued Smad-specific gene transcription in the presence of cAMP suggesting that sequestration of limited amounts of CBP/p300 by the activated cAMP/CREB pathway is the molecular basis of this inhibitory effect. These findings were extended by two functional assays. Increased intracellular cAMP levels suppressed the inductive activity of TGF-beta to contract mechanically unloaded collagen lattices and resulted in an attenuation of fibroblast migration of mechanically induced cell layer wounds. Of note, cAMP and TGF-beta synergistically induced hyaluronan synthase 2 (HAS2) expression and hyaluronan secretion, presumably via putative CREB-binding sites adjacent to Smad-binding sites within the HAS2 promoter. Our findings identify the cAMP pathway as a potent but differential and promoter-specific regulator of TGF-beta-mediated effects involved in extracellular matrix homeostasis.


Assuntos
AMP Cíclico/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Adenilil Ciclases/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Colágeno/biossíntese , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Derme/citologia , Proteína p300 Associada a E1A/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ácido Hialurônico/biossíntese , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Inibidores de Proteases/metabolismo , Transporte Proteico/efeitos dos fármacos , Elementos de Resposta/genética , Transdução de Sinais/efeitos dos fármacos
7.
Mol Cancer ; 10(1): 2, 2011 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-21211030

RESUMO

BACKGROUND: SKI and SnoN proteins have been shown to inhibit TGF-ß signaling, acting both as transcriptional co-repressors in the cell nucleus, and as sequestrators of SMAD proteins in the cytoplasm. TGF-ß, on the other hand, induces rapid, proteasome-mediated, degradation of both proteins. How elevated SKI and SnoN protein levels co-exist with active autocrine TGF-ß signaling in cancer cells is yet to be understood. RESULTS: In this study, we found elevated SKI and SnoN protein levels in a panel of melanoma cell lines, as compared to normal melanocytes. There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice. Nor did we find a correlation between SKI expression and histopathological staging of human melanoma. TGF-ß induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity. SKI knockdown in 1205Lu melanoma cells did not alter their invasive capacity or transcriptional responses to TGF-ß, and did not allow p21 expression in response to TGF-ß or reveal any growth inhibitory activity of TGF-ß. CONCLUSIONS: Despite high expression in melanoma cells, the role of SKI in melanoma remains elusive: SKI does not efficiently interfere with the pro-oncogenic activities of TGF-ß, unless stabilized by proteasome blockade. Its highly labile nature makes it an unlikely target for therapeutic intervention.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanoma/patologia , Proteínas Proto-Oncogênicas/metabolismo , Neoplasias Cutâneas/patologia , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Humanos , Leupeptinas/farmacologia , Melanoma/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Inibidores de Proteassoma , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , Neoplasias Cutâneas/metabolismo , Ativação Transcricional , Regulação para Cima
8.
J Biol Chem ; 284(46): 31523-31, 2009 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-19797115

RESUMO

GLI2 (GLI-Kruppel family member 2), a zinc finger transcription factor that mediates Hedgehog signaling, is implicated in the progression of an ever-growing number of human malignancies, including prostate and pancreatic cancer, as well as basal cell carcinoma of the skin. Its expression is up-regulated by transforming growth factor-beta (TGF-beta) in a variety of cell types, both normal and transformed. We report herein that TGF-beta-driven GLI2 expression is transcriptional and does not result from stabilization of GLI2 transcripts. We describe the characterization of the 5'-flanking sequence of human GLI2 mRNA, the identification of a transcription start site, the cloning of approximately 1,600 bp of the regulatory promoter region and the identification and functional analysis of a TGF-beta-responsive region mapped to a 91-bp sequence between nucleotides -119 and -29 of the promoter. This region harbors SMAD and lymphoid enhancer factor/T cell factor binding sites that allow functional cooperation between SMAD3 and beta-catenin, recruited to the promoter in response to TGF-beta to drive GLI2 gene transcription.


Assuntos
Fatores de Transcrição Kruppel-Like/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Proteína Smad3/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador beta/metabolismo , beta Catenina/metabolismo , Sequência de Bases , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Células Cultivadas , Imunoprecipitação da Cromatina , Clonagem Molecular , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Luciferases/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad3/genética , Transativadores/genética , Transativadores/metabolismo , Transfecção , Fator de Crescimento Transformador beta/genética , Proteína Gli2 com Dedos de Zinco , beta Catenina/genética
9.
Exp Dermatol ; 19(3): 259-68, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19765057

RESUMO

The stratum corneum (SC) is a superficial skin compartment that protects the body from the outside environment. Any disturbance of this function induces cascading steps of molecular and cellular repair in the whole epidermis. The aim of this study was to investigate epidermal gene expression following SC removal by tape stripping. Twenty-nine healthy male volunteers were included (27 +/- 4 years old). Tape stripping was processed on one inner forearm, the other unstripped forearm served as a control. Epidermis samples were collected at 2, 6, 19, 30 and 72 h after tape stripping. Trans-epidermal water loss measurements were performed at each step to monitor barrier restoration. Total RNA was extracted from collected epidermis samples and analysed by using DermArray cDNA microarrays. Among 4000 genes under investigation, we found that the expression of 370 genes varied significantly at least once during the time following stripping. Using an original clustering method, the modulated genes were gathered into eight groups. A functional characterization of the clusters enabled us to get a dynamic and global view of the main molecular processes taking place during epidermal recovery.


Assuntos
Epiderme/lesões , Epiderme/metabolismo , Expressão Gênica , Cicatrização/genética , Adulto , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Genômica , Humanos , Cinética , Masculino , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Adulto Jovem
10.
Sci Rep ; 10(1): 14491, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879407

RESUMO

GLI1 expression is broadly accepted as a marker of Hedgehog pathway activation in tumors. Efficacy of Hedgehog inhibitors is essentially limited to tumors bearing activating mutations of the pathway. GLI2, a critical Hedgehog effector, is necessary for GLI1 expression and is a direct transcriptional target of TGF-ß/SMAD signaling. We examined the expression correlations of GLI1/2 with TGFB and HH genes in 152 distinct transcriptome datasets totaling over 23,500 patients and representing 37 types of neoplasms. Their prognostic value was measured in over 15,000 clinically annotated tumor samples from 26 tumor types. In most tumor types, GLI1 and GLI2 follow a similar pattern of expression and are equally correlated with HH and TGFB genes. However, GLI1/2 broadly share prognostic value with TGFB genes and a mesenchymal/EMT signature, not with HH genes. Our results provide a likely explanation for the frequent failure of anti-Hedgehog therapies in tumors, as they suggest a key role for TGF-ß, not Hedgehog, ligands, in tumors with elevated GLI1/2-expression.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Neoplasias/diagnóstico , Proteínas Nucleares/genética , Fator de Crescimento Transformador beta/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/genética , Biologia Computacional , Perfilação da Expressão Gênica , Humanos , Ligantes , Análise Multivariada , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Risco , Transdução de Sinais/genética , Transcriptoma
11.
Mol Carcinog ; 48(6): 532-44, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18973190

RESUMO

Transforming growth factor beta (TGF-beta) exerts an important role in the late steps of carcinogenesis by cooperating with Ras to induce cell motility and tumor invasion. The transcription complex AP-1 has been implicated in the regulation of genes involved in motility and invasion, by mechanisms not yet delineated. We utilized a model of immortalized human hepatocytes (IHH) overexpressing c-Fos (IHH-Fos) or not (IHH-C) to investigate the role of c-Fos on cell motility in response to a prolonged treatment with TGF-beta, EGF or a combination of both. Cotreatment with EGF and TGF-beta, but neither cytokine alone, induced the conversion of hepatocytes to a fibroblastoid phenotype and increased their motility in Boyden chambers. EGF/TGF-beta cotreatment induced a higher effect on ERK phosphorylation compared to TGF-beta treatment alone. It also induced an increase in total and phosphorylated Ser(178) paxillin, a protein previously implicated in cell motility. This response was inhibited by two specific MEK inhibitors, indicating the involvement of the ERK pathway in paxillin activation. Overexpression of c-Fos correlated with increased cell scattering and motility, higher levels of ERK activation and phospho Ser(178) paxillin, increased levels of EGF receptor (EGF-R) mRNA and higher EGF-R phosphorylation levels following EGF/TGF-beta cotreatment. Conversely, siRNA-mediated invalidation of c-Fos delayed the appearance of fibroblastoid cells, decreased EGF-R mRNA and downregulated ERK and Ser(178) paxillin phosphorylations, indicating that c-Fos activates hepatocyte motility through an EGF-R/ERK/paxillin pathway. Since c-Fos is frequently overexpressed in hepatocarcinomas, this newly identified mechanism might be involved in the progression of hepatic tumors in vivo.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hepatócitos/citologia , Paxilina/metabolismo , Proteínas Proto-Oncogênicas c-fos/fisiologia , Serina/metabolismo , Regulação para Cima , Sequência de Bases , Linhagem Celular Transformada , Primers do DNA , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Fibroblastos/citologia , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Paxilina/química , Fosforilação , Reação em Cadeia da Polimerase , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia
12.
Cancer Res ; 67(14): 6981-6, 2007 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-17638910

RESUMO

Hedgehog (Hh) and transforming growth factor-beta (TGF-beta) family members are involved in numerous overlapping processes during embryonic development, hair cycle, and cancer. Herein, we show that TGF-beta induces the expression of the Hh signaling molecules Gli1 and Gli2 in various human cell types, including normal fibroblasts and keratinocytes, as well as various cancer cell lines. Gli2 induction by TGF-beta is rapid, independent from Hh receptor signaling, and requires a functional Smad pathway. Gli1 expression is subsequently activated in a Gli2-dependent manner. In transgenic mice overexpressing TGF-beta1 in the skin, Gli1 and Gli2 expression is also elevated and depends on Smad3. In pancreatic adenocarcinoma cell lines resistant to Hh inhibition, pharmacologic blockade of TGF-beta signaling leads to repression of cell proliferation accompanied with a reduction in Gli2 expression. We thus identify TGF-beta as a potent transcriptional inducer of Gli transcription factors. Targeting the cooperation of Hh and TGF-beta signaling may provide new therapeutic opportunities for cancer treatment.


Assuntos
Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Nucleares/metabolismo , Proteína Smad3/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Transdução de Sinais , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco
13.
Cancer Res ; 67(5): 2317-24, 2007 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17332363

RESUMO

Melanoma has a propensity to metastasize to bone, where it is exposed to high concentrations of transforming growth factor-beta (TGF-beta). Because TGF-beta promotes bone metastases from other solid tumors, such as breast cancer, we tested the role of TGF-beta in melanoma metastases to bone. 1205Lu melanoma cells, stably transfected to overexpress the natural TGF-beta/Smad signaling inhibitor Smad7, were studied in an experimental model of bone metastasis whereby tumor cells are inoculated into the left cardiac ventricle of nude mice. All mice bearing parental and mock-transfected 1205Lu cells developed osteolytic bone metastases 5 weeks post-tumor inoculation. Mice bearing 1205Lu-Smad7 tumors had significantly less osteolysis on radiographs and longer survival compared with parental and mock-transfected 1205Lu mice. To determine if the reduced bone metastases observed in mice bearing 1205Lu-Smad7 clones was due to reduced expression of TGF-beta target genes known to enhance metastases to bone from breast cancer cells, we analyzed gene expression of osteolytic factors, parathyroid hormone-related protein (PTHrP) and interleukin-11 (IL-11), the chemotactic receptor CXCR4, and osteopontin in 1205Lu cells. Quantitative reverse transcription-PCR analysis indicated that PTHrP, IL-11, CXCR4, and osteopontin mRNA steady-state levels were robustly increased in response to TGF-beta and that Smad7 and the TbetaRI small-molecule inhibitor, SB431542, prevented such induction. In addition, 1205Lu-Smad7 bone metastases expressed significantly lower levels of IL-11, connective tissue growth factor, and PTHrP. These data suggest that TGF-beta promotes osteolytic bone metastases due to melanoma by stimulating the expression of prometastatic factors via the Smad pathway. Blockade of TGF-beta signaling may be an effective treatment for melanoma metastasis to bone.


Assuntos
Neoplasias Ósseas/secundário , Melanoma/genética , Melanoma/patologia , Proteína Smad7/genética , Animais , Neoplasias Ósseas/genética , Feminino , Humanos , Melanoma/metabolismo , Camundongos , Camundongos Nus , Metástase Neoplásica/genética , Proteína Smad7/metabolismo , Análise de Sobrevida , Transfecção , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas
14.
J Dermatol Sci ; 94(3): 321-329, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31208857

RESUMO

BACKGROUND: Tyrosinase-Related Protein 2 (TRP2) is an enzyme involved in melanogenesis, that also exerts proliferative, anti-apoptotic and immunogenic functions in melanoma cells. TRP2 transcription is regulated by the melanocytic master transcription factor MITF. GLI2, a transcription factor that acts downstream of Hedgehog signaling, is also a direct transcriptional target of the TGF-ß/SMAD pathway that contributes to melanoma progression and exerts transcriptional antagonistic activities against MITF. OBJECTIVES: To characterize the molecular events responsible for TGF-ß and GLI2 repression of TRP2 expression. METHODS: In silico promoter analysis, transient cell transfection experiments with 5'-end TRP2 promoter deletion constructs, chromatin immuno-precipitation, and site-directed promoter mutagenesis were used to dissect the molecular mechanisms of TRP2 gene regulation by TGF-ß and GLI2. RESULTS: We demonstrate that TGF-ß and GLI2-specific TRP2 repression involves direct mechanisms that occur in addition to MITF downregulation by TGF-ß and GLI2. We identify two functional GLI2 binding sites within the TRP2 promoter that are critical for TGF-ß and GLI2 responsiveness, one of them overlapping a CREB binding site. GLI2 and CREB competing for the same cis-element is associated with opposite transcriptional outcome. CONCLUSION: Our results further refine the understanding of how TGF-ß and GLI2 control the phenotypic plasticity of melanoma cells. In particular, we identify critical GLI2-binding cis-elements within the TRP2 promoter region that allow for its transcriptional repression independently from MITF concomitant downregulation.


Assuntos
Regulação Neoplásica da Expressão Gênica , Oxirredutases Intramoleculares/genética , Melanoma/genética , Proteínas Nucleares/metabolismo , Neoplasias Cutâneas/genética , Fator de Crescimento Transformador beta/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismo , Linhagem Celular Tumoral , Biologia Computacional , Humanos , Melanoma/patologia , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas/genética , Neoplasias Cutâneas/patologia , Transcrição Gênica
15.
Carcinogenesis ; 29(3): 536-43, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18174238

RESUMO

Nuclear factor kappaB (NF-kappaB) and activator protein 1 are transcription factors involved in the regulation of cell proliferation that play important roles in tumorigenesis. We investigated whether these two factors cooperate for transcriptional regulation of cyclin D1 (CCND1), a gene whose deregulation is critical during carcinogenesis. We demonstrate that overexpression of JunD in human hepatocarcinoma cells strongly activates transcription mediated by the kappaB2 site of the CCND1 promoter in reporter assays, in a manner strictly dependent on the presence of NF-kappaB proteins. Serum stimulation increased the expression of p65, p50, c-Fos, c-Jun and JunD and induced the recruitment of p65, p50 and JunD to the kappaB2 site of the promoter in DNA pull-down assays. Chromatin immunoprecipitation (ChIP) analysis confirmed the serum-induced recruitment of JunD to the promoter in vivo and showed that the presence of JunD was dependent on the presence of p65 and p50, indicating a protein-protein-dependent mechanism of JunD recruitment. Serum-induced activation of protein binding to kappaB2 correlated with high levels of phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylation. Both LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), and overexpression of a dominant-negative form of PDK-1 inhibited the JunD-stimulating effect in reporter assays. LY294002 also prevented the serum-induced recruitment of JunD, but not p65 or p50 to the promoter in ChIP assay. JunD-p65 complexes, identified in vivo by co-immunoprecipitation, were decreased by LY294002 and by small interfering RNA inhibition of PDK-1. Taken together, our data demonstrate a PI3K/PDK-1-dependent functional cooperation of NF-kappaB and JunD in the transcriptional regulation of CCND1 by serum.


Assuntos
Ciclina D1/genética , Fosfatidilinositol 3-Quinases/fisiologia , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Fator de Transcrição RelA/fisiologia , Sequência de Bases , Sítios de Ligação , Sangue , Imunoprecipitação da Cromatina , Cromonas/farmacologia , Ciclina D1/química , Ciclina D1/metabolismo , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Inibidores Enzimáticos/farmacologia , Humanos , Imunoprecipitação , Morfolinas/farmacologia , Piruvato Desidrogenase Quinase de Transferência de Acetil , Fator de Transcrição RelA/metabolismo
16.
J Cell Physiol ; 216(2): 438-44, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18297686

RESUMO

We used both pharmacological and gene knockout approaches to elucidate the specific roles played by the Jun-N-terminal kinase (JNK) and NFkappaB pathways downstream of TNF-alpha in the context of connexin43 (Cx43) gene expression. We demonstrate that TNF-alpha reduces the expression of Cx43 in HaCat cell lines at the protein and mRNA levels, and transcriptionally. We also demonstrate that TNF-alpha decreases gap junctional intercellular communication (GJIC) between HaCat cells. Using pharmacological inhibitors of the NFkappaB signaling pathway, we determined that the NFkappaB signaling cascade is not implicated in TNF-alpha effect on Cx43 expression and in the subsequent decrease of GJIC. Conversely, in NFkappaB essential modulator (NEMO(-)) fibroblasts, lack of NFkappaB activation did not influence both the effect of TNF-alpha on Cx43 expression and on GJIC. In contrast, pharmacologic inhibition of JNK abolishes TNF-alpha-driven repression of Cx43 gene expression and GJIC between HaCat cells. Using JNK(1) (-/-)-JNK(2) (-/-) (JNK(-/-)) fibroblasts, we demonstrate that similar regulatory mechanisms take place in fibroblasts. Together, these results identify JNK and not NFkappaB, as a critical mediator of TNF-alpha repressory effect on Cx43 gene expression.


Assuntos
Conexina 43/metabolismo , Regulação da Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Queratinócitos/fisiologia , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Comunicação Celular/fisiologia , Linhagem Celular , Conexina 43/genética , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/fisiologia , Junções Comunicantes/metabolismo , Marcação de Genes , Genes Reporter , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Queratinócitos/citologia , NF-kappa B/genética , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/genética
17.
J Cell Physiol ; 217(3): 759-68, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18668519

RESUMO

One of the shared physiological roles between TGF-beta and connexin family members is to inhibit epithelial cell cycle progression and consequently, to provide protection against malignant transformation. Herein, we demonstrated that TGF-beta1 induces the expression of connexin43 (Cx43) in normal murine mammary gland (NMuMG) cell lines at the protein and mRNA levels, and transcriptionally. Using overexpression of a truncated dominant-negative form of Cx43, we determined that the modulation of gap junctional communication by TGF-beta1 plays a key role in the control of NMuMG cells proliferation by TGF-beta1. In addition, using overexpression of truncated dominant-negative forms of either Smad2 or Smad3, and MDA-MB-468 human breast carcinoma cells deficient for Smad4, we determined that the Smad cascade is not implicated in TGF-beta1 effect on Cx43 expression. Using specific pharmacologic inhibitors for JNK, ERK, p38, and PI3K/AKT signaling pathways, we demonstrated the cooperative role of p38 and PI3K/AKT signaling in TGF-beta1-induced Cx43 expression and gap junctional communication. Furthermore, transfection of a c-jun antisense expression vector significantly prevented TGF-beta1-induced Cx43 gene expression demonstrating the involvement of c-Jun/AP-1 pathway together with p38 and PI3K/AKT pathways in mediating TGF-beta1-induced Cx43 gene expression.


Assuntos
Conexina 43/genética , Células Epiteliais/enzimologia , Glândulas Mamárias Animais/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/enzimologia , Humanos , Glândulas Mamárias Animais/enzimologia , Camundongos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Xenopus
18.
World J Gastroenterol ; 14(41): 6339-46, 2008 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-19009649

RESUMO

AIM: To investigate the effect of stable c-Fos overexpression on immortalized human hepatocyte (IHH) proliferation. METHODS: IHHs stably transfected with c-Fos (IHH-Fos) or an empty vector (IHH-C) were grown in medium supplemented with 1% serum or stimulated with 10% serum. Cell proliferation was assessed by cell counts, 3H-thymidine uptake and flow cytometry analyses. The levels of cell cycle regulatory proteins (Cyclin D1, E, A) cyclin dependent kinases (cdk) cdk2, cdk4, cdk6, and their inhibitors p15, p16, p21, p27, total and phosphorylated GSK-3beta and epidermal growth factor receptor (EGF-R) were assayed by Western blotting. Analysis of Cyclin D1 mRNA levels was performed by reverse transcription-polymerase chain reaction and real-time polymerase chain reaction (PCR) analysis. Stability of Cyclin D1 was studied by cycloheximide blockade experiments. RESULTS: Stable c-Fos overexpression increased cell proliferation under low serum conditions and resulted in a two-fold increase in [3H]-thymidine incorporation following serum addition. Cell cycle analysis by flow cytometry showed that c-Fos accelerated the cell cycle kinetics. Following serum stimulation, Cyclin D1 was more abundantly expressed in c-Fos overexpressing cells. Cyclin D1 accumulation did not result from increased transcriptional activation, but from nuclear stabilization. Overexpression of c-Fos correlated with higher nuclear levels of inactive phosphorylated GSK-3beta, a kinase involved in Cyclin D1 degradation and higher levels of EGF-R mRNA, and EGF-R protein compared to IHH-C both in serum starved, and in serum stimulated cells. Abrogation of EGF-R signalling in IHH-Fos by treatment with AG1478, a specific EGF-R tyrosine kinase inhibitor, prevented the phosphorylation of GSK-3beta induced by serum stimulation and decreased Cyclin D1 stability in the nucleus. CONCLUSION: Our results clearly indicate a positive role for c-Fos in cell cycle regulation in hepatocytes. Importantly, we delineate a new mechanism by which c-Fos could contribute to hepatocarcinogenesis through stabilization of Cyclin D1 within the nucleus, evoking a new feature to c-Fos implication in hepatocellular carcinoma.


Assuntos
Núcleo Celular/metabolismo , Proliferação de Células , Ciclina D1/metabolismo , Hepatócitos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ciclo Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Cicloeximida/farmacologia , Receptores ErbB/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Cinética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Estabilidade Proteica , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-fos/genética , Quinazolinas , RNA Mensageiro/metabolismo , Transfecção , Tirfostinas/farmacologia , Regulação para Cima
19.
Int J Biochem Cell Biol ; 98: 75-81, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29544895

RESUMO

The ubiquitous distribution of both Hippo and TGF-ß signaling cascade components and their critical implication in tissue homeostasis and disease has led to the discovery of a remarkable slew of interesting and unique features regarding their functional crosstalks. Upstream cellular cues regulating the Hippo pathway, including cell-cell contacts and apico-basal cell polarity have been well characterized. Herein, we provide an overview of the published models of compartmentalized signaling crosstalk mechanisms between Hippo signaling and the TGF-ß/SMAD pathway. How cell polarity impacts the interaction between the two pathways is discussed, together with the specifics of cytoplasmic and nuclear events implicating SMADs and YAP/TAZ, leading to contextual regulation of target gene expression.


Assuntos
Polaridade Celular , Células Epiteliais/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células Epiteliais/citologia , Via de Sinalização Hippo , Humanos , Transdução de Sinais
20.
Eur J Pharmacol ; 573(1-3): 65-9, 2007 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-17706637

RESUMO

Halofuginone, an alkaloid isolated from the plant Dichroa febrifuga, has been shown to be a potent inhibitor of tissue fibrosis. We herein demonstrate that, at concentrations below 10(-7) M, halofuginone does not affect the cell cycle but efficiently induces extracellular signal-regulated kinases(1,2) (ERK(1,2)), p38 and Jun NH2-terminal kinases(1,2) (JNK(1,2)) phosphorylation. In addition, at these non cytotoxic concentrations, halofuginone diminishes the capacity of fibroblasts to contract mechanically unloaded collagen lattices, an effect that is specifically blocked by the ERK inhibitors PD98059 and U0126, not by inhibitors of the JNK or p38 pathways. These data thus indicate that the inhibitory effect of halofuginone on fibroblast contractile activity, a key function for wound healing implicated in the development of tissue fibrosis, is an ERK-mediated mechanism.


Assuntos
Colágeno Tipo I/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Piperidinas/farmacologia , Quinazolinonas/farmacologia , Western Blotting , Butadienos/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Fibroblastos/citologia , Fibroblastos/metabolismo , Flavonoides/farmacologia , Humanos , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Piridinas/farmacologia , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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