RESUMO
Carcinoma-associated fibroblasts (CAFs) play an important role in the progression of multiple malignancies. Secretion of cytokines and growth factors underlies the pro-tumoral effect of CAFs. Although this paracrine function has been extensively documented, the molecular mechanisms controlling the expression of these factors remain elusive. In this study, we provide evidence of a novel CAF transcriptional axis regulating the expression of SDF1, a major driver of cancer cell migration, involving the transcription factor GLI1 and histone acetyltransferase p300. We demonstrate that conditioned media from CAFs overexpressing GLI1 induce the migration of pancreatic cancer cells, and this effect is impaired by an SDF1-neutralizing antibody. Using a combination of co-immunoprecipitation, proximity ligation assay and chromatin immunoprecipitation assay, we further demonstrate that GLI1 and p300 physically interact in CAFs to co-occupy and drive SDF1 promoter activity. Mapping experiments highlight the requirement of GLI1 N-terminal for the interaction with p300. Importantly, knockdowns of both GLI1 and p300 reduce SDF1 expression. Further analysis shows that knockdown of GLI1 decreases SDF1 promoter activity, p300 recruitment, and levels of its associated histone marks (H4ac, H3K27ac, and H3K14ac). Finally, we show that the integrity of two GLI binding sites in the SDF1 promoter is required for p300 recruitment. Our findings define a new role for the p300-GLI1 complex in the regulation of SDF1, providing new mechanistic insight into the molecular events controlling pancreatic cancer cells migration.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Pancreáticas , Humanos , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Movimento Celular , Imunoprecipitação da Cromatina , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Quimiocina CXCL12/metabolismo , Neoplasias PancreáticasRESUMO
Germline mutations in CDKN2A, encoding the tumor suppressor p16, are responsible for a large proportion of familial melanoma cases and also increase risk of pancreatic cancer. We identified four families through pancreatic cancer probands that were affected by both cancers. These families bore a germline missense variant of CDKN2A (47T>G), encoding a p16-L16R mutant protein associated with high cancer occurrence. Here, we investigated the biological significance of this variant. When transfected into p16-null pancreatic cancer cells, p16-L16R was expressed at lower levels than wild-type (WT) p16. In addition, p16-L16R was unable to bind CDK4 or CDK6 compared with WT p16, as shown by coimmunoprecipitation assays and also was impaired in its ability to inhibit the cell cycle, as demonstrated by flow cytometry analyses. In silico molecular modeling predicted that the L16R mutation prevents normal protein folding, consistent with the observed reduction in expression/stability and diminished function of this mutant protein. We isolated normal dermal fibroblasts from members of the families expressing WT or L16R proteins to investigate the impact of endogenous p16-L16R mutant protein on cell growth. In culture, p16-L16R fibroblasts grew at a faster rate, and most survived until later passages than p16-WT fibroblasts. Further, western blotting demonstrated that p16 protein was detected at lower levels in p16-L16R than in p16-WT fibroblasts. Together, these results suggest that the presence of a CDKN2A (47T>G) mutant allele contributes to an increased risk of pancreatic cancer as a result of reduced p16 protein levels and diminished p16 tumor suppressor function.
Assuntos
Ciclo Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Heterozigoto , Melanoma/patologia , Neoplasias Pancreáticas/patologia , Adulto , Idoso , Feminino , Humanos , Masculino , Melanoma/genética , Pessoa de Meia-Idade , Neoplasias Pancreáticas/genética , LinhagemRESUMO
The modulation of GLI2, an oncogenic transcription factor commonly upregulated in cancer, is in many cases not due to genetic defects, suggesting dysregulation through alternative mechanisms. The identity of these molecular events remains for the most part unknown. Here, we identified TFII-I as a novel repressor of GLI2 expression. Mapping experiments suggest that the INR region of the GLI2 promoter is necessary for GLI2 repression. ChIP studies showed that TFII-I binds to this INR. TFII-I knockdown decreased the binding of NELF-A, a component of the promoter-proximal pausing complex at this site, and enriched phosphorylated RNAPII serine 2 in the GLI2 gene body. Immunoprecipitation studies demonstrate TFII-I interaction with SPT5, another pausing complex component. TFII-I overexpression antagonized GLI2 induction by TGFß, a known activator of GLI2 in cancer cells. TGFß reduced endogenous TFII-I binding to the INR and increased RNAPII SerP2 in the gene body. We demonstrate that this regulatory mechanism is not exclusive of GLI2. TGFß-induced genes CCR7, TGFß1 and EGR3 showed similar decreased TFII-I and NELF-A INR binding and increased RNAPII SerP2 in the gene body post-TGFß treatment. Together these results identify TFII-I as a novel repressor of a subset of TGFß-responsive genes through the regulation of RNAPII pausing.
Assuntos
Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição TFII/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismo , Células Hep G2 , Humanos , Regiões Promotoras Genéticas , Proteínas Repressoras/fisiologia , Transcrição Gênica , Ativação TranscricionalRESUMO
The transcription factor GLI1 (GLI family zinc finger 1) plays a key role in the development and progression of multiple malignancies. To date, regulation of transcriptional activity at target gene promoters is the only molecular event known to underlie the oncogenic function of GLI1. Here, we provide evidence that GLI1 controls chromatin accessibility at distal regulatory regions by modulating the recruitment of SMARCA2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2) to these elements. We demonstrate that SMARCA2 endogenously interacts with GLI1 and enhances its transcriptional activity. Mapping experiments indicated that the C-terminal transcriptional activation domain of GLI1 and SMARCA2's central domains, including its ATPase motif, are required for this interaction. Interestingly, similar to SMARCA2, GLI1 overexpression increased chromatin accessibility, as indicated by results of the micrococcal nuclease assay. Further, results of assays for transposase-accessible chromatin with sequencing (ATAC-seq) after GLI1 knockdown supported these findings, revealing that GLI1 regulates chromatin accessibility at several regions distal to gene promoters. Integrated RNA-seq and ATAC-seq data analyses identified a subset of differentially expressed genes located in cis to these regulated chromatin sites. Finally, using the GLI1-regulated gene HHIP (Hedgehog-interacting protein) as a model, we demonstrate that GLI1 and SMARCA2 co-occupy a distal chromatin peak and that SMARCA2 recruitment to this HHIP putative enhancer requires intact GLI1. These findings provide insights into how GLI1 controls gene expression in cancer cells and may inform approaches targeting this oncogenic transcription factor to manage malignancies.
Assuntos
Cromatina/genética , Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Proteína GLI1 em Dedos de Zinco/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Células HEK293 , Humanos , Domínios Proteicos , Mapas de Interação de Proteínas , Fatores de Transcrição/química , Ativação Transcricional , Proteína GLI1 em Dedos de Zinco/químicaRESUMO
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/genéticaRESUMO
Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFAs). However, the molecular mechanisms modulating this phenomenon are in part poorly understood. Here, we provide evidence of a novel antitumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreased tumor growth and metastasis and increased apoptosis. Molecular analysis of this effect showed significantly reduced expression of a subset of antiapoptotic proteins, including BCL2, BFL1/A1, and 4-1BB, in AA-treated cells. We demonstrated that down-regulation of the transcription factor glioma-associated protein 1 (GLI1) in AA-treated cells is the underlying mechanism controlling BCL2, BFL1/A1, and 4-1BB expression. Using luciferase reporters, chromatin immunoprecipitation, and expression studies, we found that GLI1 binds to the promoter of these antiapoptotic molecules and regulates their expression and promoter activity. We provide evidence that AA-induced apoptosis and down-regulation of antiapoptotic genes can be inhibited by overexpressing GLI1 in AA-sensitive cells. Conversely, inhibition of GLI1 mimics AA treatments, leading to decreased tumor growth, cell viability, and expression of antiapoptotic molecules. Further characterization showed that AA represses GLI1 expression by stimulating nuclear translocation of NFATc1, which then binds the GLI1 promoter and represses its transcription. AA was shown to increase reactive oxygen species. Treatment with antioxidants impaired the AA-induced apoptosis and down-regulation of GLI1 and NFATc1 activation, indicating that NFATc1 activation and GLI1 repression require the generation of reactive oxygen species. Collectively, these results define a novel mechanism underlying AA antitumoral functions that may serve as a foundation for future PUFA-based therapeutic approaches.
Assuntos
Ácido Araquidônico/farmacologia , Proliferação de Células/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Neoplasias/metabolismo , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral , Puffs Cromossômicos , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Fatores de Transcrição NFATC/genética , Neoplasias/genética , Neoplasias/fisiopatologia , Regiões Promotoras Genéticas , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de ZincoRESUMO
Ig secretion by terminally differentiated B cells is an important component of the immune response to foreign pathogens. Its overproduction is a defining characteristic of several B cell malignancies, including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Therefore, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for this disease. In this study, we define a novel pathway involving the oncogenic transcription factor GLI2 modulating IgM secretion by WM malignant cells. Pharmacological and genetic inhibition of GLI2 in WM malignant cells resulted in a reduction in IgM secretion. Screening for a mechanism identified the IL-6Rα (gp80) subunit as a downstream target of GLI2 mediating the regulation of IgM secretion. Using a combination of expression, luciferase, and chromatin immunoprecipitation assays we demonstrate that GLI2 binds to the IL-6Rα promoter and regulates its activity as well as the expression of this receptor. Additionally, we were able to rescue the reduction in IgM secretion in the GLI2 knockdown group by overexpressing IL-6Rα, thus defining the functional significance of this receptor in GLI2-mediated regulation of IgM secretion. Interestingly, this occurred independent of Hedgehog signaling, a known regulator of GLI2, as manipulation of Hedgehog had no effect on IgM secretion. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.
Assuntos
Linfócitos B/imunologia , Imunoglobulina M/imunologia , Fatores de Transcrição Kruppel-Like/imunologia , Receptores de Interleucina-6/imunologia , Macroglobulinemia de Waldenstrom/patologia , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Feminino , Proteínas Hedgehog/genética , Humanos , Receptores de Hialuronatos/imunologia , Imunoglobulina M/biossíntese , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas/genética , Ligação Proteica/imunologia , Receptores de Interleucina-6/biossíntese , Transdução de Sinais/imunologia , Macroglobulinemia de Waldenstrom/metabolismo , Proteína Gli2 com Dedos de ZincoRESUMO
The biological role of the transcription factor GLI1 in the regulation of tumor growth is well established; however, the molecular events modulating this phenomenon remain elusive. Here, we demonstrate a novel mechanism underlying the role of GLI1 as an effector of TGFß signaling in the regulation of gene expression in cancer cells. TGFß stimulates GLI1 activity in cancer cells and requires its transcriptional activity to induce BCL2 expression. Analysis of the mechanism regulating this interplay identified a new transcriptional complex including GLI1 and the TGFß-regulated transcription factor, SMAD4. We demonstrate that SMAD4 physically interacts with GLI1 for concerted regulation of gene expression and cellular survival. Activation of the TGFß pathway induces GLI1-SMAD4 complex binding to the BCL2 promoter whereas disruption of the complex through SMAD4 RNAi depletion impairs GLI1-mediated transcription of BCL2 and cellular survival. Further characterization demonstrated that SMAD2 and the histone acetyltransferase, PCAF, participate in this regulatory mechanism. Both proteins bind to the BCL2 promoter and are required for TGFß- and GLI1-stimulated gene expression. Moreover, SMAD2/4 RNAi experiments showed that these factors are required for the recruitment of GLI1 to the BCL2 promoter. Finally, we determined whether this novel GLI1 transcriptional pathway could regulate other TGFß targets. We found that two additional TGFß-stimulated genes, INTERLEUKIN-7 and CYCLIN D1, are dependent upon the intact GLI1-SMAD-PCAF complex for transcriptional activation. Collectively, these results define a novel epigenetic mechanism that uses the transcription factor GLI1 and its associated complex as a central effector to regulate gene expression in cancer cells.
Assuntos
Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Proteína Smad4/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Epigênese Genética/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/fisiologia , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad4/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/genética , Proteína GLI1 em Dedos de ZincoRESUMO
Tissue regeneration requires the activation of a set of specific growth signaling pathways. The identity of these cascades and their biological roles are known; however, the molecular mechanisms regulating the interplay between these pathways remain poorly understood. Here, we define a new role for SULFATASE 2 (SULF2) in regulating tissue regeneration and define the WNT-GLI1 axis as a novel downstream effector for this sulfatase in a liver model of tissue regeneration. SULF2 is a heparan sulfate 6-O-endosulfatase, which releases growth factors from extracellular storage sites turning active multiple signaling pathways. We demonstrate that SULF2-KO mice display delayed regeneration after partial hepatectomy (PH). Mechanistic analysis of the SULF2-KO phenotype showed a decrease in WNT signaling pathway activity in vivo. In isolated hepatocytes, SULF2 deficiency blocked WNT-induced ß-CATENIN nuclear translocation, TCF activation, and proliferation. Furthermore, we identified the transcription factor GLI1 as a novel target of the SULF2-WNT cascade. WNT induces GLI1 expression in a SULF2- and ß-CATENIN-dependent manner. GLI1-KO mice phenocopied the SULF2-KO, showing delayed regeneration and decreased hepatocyte proliferation. Moreover, we identified CYCLIN D1, a key mediator of cell growth during tissue regeneration, as a GLI1 transcriptional target. GLI1 binds to the cyclin d1 promoter and regulates its activity and expression. Finally, restoring GLI1 expression in the liver of SULF2-KO mice after PH rescues CYCLIN D1 expression and hepatocyte proliferation to wild-type levels. Thus, together these findings define a novel pathway in which SULF2 regulates tissue regeneration in part via the activation of a novel WNT-GLI1-CYCLIN D1 pathway.
Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Regeneração Hepática , Sulfatases/metabolismo , Via de Sinalização Wnt , Animais , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Hepatectomia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Regeneração Hepática/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Sulfatases/deficiência , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3A/farmacologia , Proteína GLI1 em Dedos de Zinco , beta Catenina/metabolismoRESUMO
Aberrant activation of GLI transcription factors has been implicated in the pathogenesis of different tumor types including pancreatic ductal adenocarcinoma. However, the mechanistic link with established drivers of this disease remains in part elusive. In this study, using a new genetically engineered mouse model overexpressing constitutively active mouse form of GLI2 and a combination of genome-wide assays, we provide evidence of a novel mechanism underlying the interplay between KRAS, a major driver of pancreatic ductal adenocarcinoma development, and GLI2 to control oncogenic gene expression. These mice, also expressing KrasG12D, show significantly reduced median survival rate and accelerated tumorigenesis compared with the KrasG12D only expressing mice. Analysis of the mechanism using RNA sequencing demonstrate higher levels of GLI2 targets, particularly tumor growth-promoting genes, including Ccnd1, N-Myc, and Bcl2, in KrasG12D mutant cells. Furthermore, chromatin immunoprecipitation sequencing studies showed that in these cells KrasG12D increases the levels of trimethylation of lysine 4 of the histone 3 (H3K4me3) at the promoter of GLI2 targets without affecting significantly the levels of other major active chromatin marks. Importantly, Gli2 knockdown reduces H3K4me3 enrichment and gene expression induced by mutant Kras. In summary, we demonstrate that Gli2 plays a significant role in pancreatic carcinogenesis by acting as a downstream effector of KrasG12D to control gene expression.
Assuntos
Carcinoma Ductal Pancreático , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Proteína Gli2 com Dedos de Zinco , Animais , Humanos , Camundongos , Carcinogênese , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Histonas/metabolismo , Histonas/genética , Camundongos Transgênicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transcrição Gênica , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismoRESUMO
PURPOSE: Genomic testing has increased the quantity of information available to oncologists. Unfortunately, many identified sequence alterations are variants of unknown significance (VUSs), which thus limit the clinician's ability to use these findings to inform treatment. We applied a combination of in silico prediction and molecular modeling tools and laboratory techniques to rapidly define actionable VUSs. MATERIALS AND METHODS: Exome sequencing was conducted on 308 tumors from various origins. Most single nucleotide alterations within gene coding regions were VUSs. These VUSs were filtered to identify a subset of therapeutically targetable genes that were predicted with in silico tools to be altered in function by their variant sequence. A subset of receptor tyrosine kinase VUSs was characterized by laboratory comparison of each VUS versus its wild-type counterpart in terms of expression and signaling activity. RESULTS: The study identified 4,327 point mutations of which 3,833 were VUSs. Filtering for mutations in genes that were therapeutically targetable and predicted to affect protein function reduced these to 522VUSs of interest, including a large number of kinases. Ten receptortyrosine kinase VUSs were selected to explore in the laboratory. Of these, seven were found to be functionally altered. Three VUSs (FGFR2 F276C, FGFR4 R78H, and KDR G539R) showed increased basal or ligand-stimulated ERK phosphorylation compared with their wild-type counterparts, which suggests that they support transformation. Treatment of a patient who carried FGFR2 F276C with an FGFR inhibitor resulted in significant and sustained tumor response with clinical benefit. CONCLUSION: The findings demonstrate the feasibility of rapid identification of the biologic relevance of somatic mutations, which thus advances clinicians' ability to make informed treatment decisions.
RESUMO
PURPOSE: A previous study by the authors has shown that recombinant myocilin purified from a prokaryotic expression system increases outflow resistance in cultured human anterior segments. The present study was performed to determine whether full-length myocilin purified from a human trabecular meshwork cell expression system alters outflow resistance after infusion into human anterior segments. METHODS: A feline immunodeficiency virus vector encoding both full-length myocilin (amino acids 1-503 fused to C-terminal V5 and six-histidine epitopes) and puromycin resistance was used to transduce a transformed trabecular meshwork cell line (TM5). Stably expressing cells were selected with puromycin. Recombinant myocilin was purified from the media using nickel ion affinity chromatography. Control purifications were performed on media from parental TM5 cells. Anterior segments of human eyes were placed in organ culture and perfused with either Dulbecco's modified Eagle's medium (DMEM) or DMEM supplemented with 50% porcine aqueous humor. One eye received an anterior chamber exchange with recombinant myocilin (2 microg/mL), whereas the fellow eye received an equal volume of control. Immunohistochemistry was performed with anti-myocilin and anti-V5 antibodies. Native polyacrylamide gel electrophoresis was used to analyze myocilin complex formation in porcine aqueous humor. RESULTS: Recombinant myocilin in porcine aqueous humor increased outflow resistance in cultured human anterior segments (91% +/- 68% [mean +/- SD] versus 18% +/- 31% in fellow control eye; n = 9, P = 0.004). Maximum outflow resistance was obtained 5 to 17 hours after infusion and remained above baseline for >3 days. Recombinant myocilin also increased outflow resistance in eyes incubated in DMEM, but only if myocilin was preincubated with porcine aqueous humor (78% +/- 77% when preincubated in DMEM containing porcine aqueous humor versus 13% +/- 15% when preincubated with DMEM alone, n = 6, P = 0.03). Recombinant myocilin appears to form a complex in porcine aqueous humor with a heat-labile protein(s). Immunohistochemistry revealed the presence of myocilin in the juxtacanalicular region of the trabecular meshwork. CONCLUSIONS: Myocilin purified from human trabecular meshwork cells increased outflow resistance in cultured human anterior segments, but only after incubation with porcine aqueous humor. Recombinant myocilin appears to form a complex in porcine aqueous humor that enables it to bind specifically within the trabecular meshwork.
Assuntos
Humor Aquoso/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Histidina/metabolismo , Malha Trabecular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular , Feminino , Expressão Gênica , Vetores Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Proteínas Recombinantes de Fusão/metabolismo , Suínos , Malha Trabecular/citologia , TransfecçãoRESUMO
PURPOSE: To determine whether aqueous humor, the in vivo source of nutrients for trabecular meshwork cells, alters cellular and molecular characteristics in primary trabecular monolayer cell cultures when compared with standard culture conditions. METHODS: Human primary trabecular meshwork cell cultures were grown in DMEM supplemented with 50% human aqueous humor (DMEM-AH), heat-denatured DMEM-AH, 10% fetal bovine serum (DMEM-FBS, the standard culture supplement), or heat-denatured DMEM-FBS. Confluent trabecular cells were assayed for cell propagation and morphology for 21 days. Protein expression profiles of trabecular cell lysates were analyzed by two-dimensional polyacrylamide gel electrophoresis. Western blot analysis was used to determine the protein expression of myocilin and TIMP-1 in conditioned media collected from trabecular cells at 5, 10, 15, and 21 days. Myocilin expression was also analyzed by Western immunoblots after addition of dexamethasone (10(-7) M) or ascorbic acid (29 mg/dL). RESULTS: Trabecular cells supplemented with DMEM-AH for 21 days showed decreased cell proliferation when compared with DMEM-FBS (11% vs. 141%). Cellular morphology was also altered: Trabecular cells incubated in DMEM-AH showed larger-, broader-, and flatter-appearing cells than did the more spindle-shaped cells grown in DMEM-FBS. Protein profiles of trabecular cell lysates isolated from cells incubated in DMEM-AH differed from those incubated in DMEM-FBS. In DMEM-AH-conditioned medium, myocilin expression was increased and TIMP-1 expression was decreased at day 21. Induction of myocilin by dexamethasone was observed in conditioned medium isolated from cells treated with DMEM-FBS (442%), but only a 10% increase in myocilin was observed beyond the normal induction in DMEM-AH. Daily administration of ascorbic acid to DMEM-AH failed to increase myocilin expression beyond that obtained with DMEM-AH. CONCLUSIONS: Addition of human aqueous humor rather than FBS to trabecular monolayer cell cultures triggers significant changes in cellular and molecular characteristics. The protein component of aqueous humor is responsible for these changes. Aqueous humor supplementation may maintain cultured trabecular cells in a more physiologic state.
Assuntos
Humor Aquoso/fisiologia , Meios de Cultura , Malha Trabecular/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacologia , Western Blotting , Técnicas de Cultura de Células , Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , Dexametasona/farmacologia , Eletroforese em Gel Bidimensional , Proteínas do Olho/metabolismo , Feminino , Glicoproteínas/metabolismo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Soro , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/metabolismoRESUMO
PURPOSE: Myocilin forms large complexes in aqueous humor. Part of this complex formation is due to myocilin-myocilin protein non-covalent interactions within the leucine zipper. However, additional covalent interactions also exist. We investigated the role of these covalent interactions in disulfide bond formation within myocilin. METHODS: Human aqueous humor was separated by denatured/non-reduced SDS-PAGE followed by Western blot analysis with myocilin specific antibodies. In part two of the study, site-directed mutagenesis was used to selectively mutate one, two, three, four, and all five cysteine residues in the mature myocilin protein expressed in an in vitro system. Products were immunoprecipitated with a hemagglutinin polyclonal antibody following in vitro transcription/translation and analyzed by SDS-PAGE. In part three of the study, glaucoma associated myocilin mutations Arg82Cys and Cys433Arg were created and complex formation analyzed in trabecular meshwork cells. RESULTS: Human aqueous humor showed myocilin in several distinct large complexes in non-reduced SDS-PAGE gels, indicating disulfide bonds occur. Similarly, in vitro expressed myocilin also produced large complexes. Mutation of all five cysteines (within the mature myocilin protein) eliminated this large complex formation. A combination of cysteine to alanine substitutions at amino acids 185, 245, and 433 had the most influence on myocilin complex formation under non-reducing conditions, however individual substitutions at each of the five cysteine amino acids had little influence on myocilin complexes. In trabecular meshwork cells, Arg82Cys was secreted but formed different sized complexes than wild type myocilin. Cys433Arg was not secreted and remained intracellular in a pattern that differed from wild type myocilin and Arg82Cys. CONCLUSIONS: Myocilin complexes present in human aqueous humor are in part due to disulfide bond formation between cysteine amino acids. Glaucoma associated mutations that affect the number of cysteine residues may alter covalent interactions.
Assuntos
Cisteína/metabolismo , Dissulfetos/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Humor Aquoso/metabolismo , Western Blotting , Células Cultivadas , Cisteína/genética , Proteínas do Citoesqueleto , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Proteínas do Olho/genética , Glaucoma/metabolismo , Glicoproteínas/genética , Humanos , Mutagênese Sítio-Dirigida , Plasmídeos , Malha Trabecular/metabolismoRESUMO
The Hedgehog (HH) pathway has been identified as an important deregulated signal transduction pathway in pancreatic ductal adenocarcinoma (PDAC), a cancer type characterized by a highly metastatic phenotype. In PDAC, the canonical HH pathway activity is restricted to the stromal compartment while HH signaling in the tumor cells is reduced as a consequence of constitutive KRAS activation. Here, we report that in the tumor compartment of PDAC the HH pathway effector transcription factor GLI1 regulates epithelial differentiation. RNAi-mediated knockdown of GLI1 abolished characteristics of epithelial differentiation, increased cell motility, and synergized with TGFß to induce an epithelial-to-mesenchymal transition (EMT). Notably, EMT conversion in PDAC cells occurred in the absence of induction of SNAIL or SLUG, two canonical inducers of EMT in many other settings. Further mechanistic analysis revealed that GLI1 directly regulated the transcription of E-cadherin, a key determinant of epithelial tissue organization. Collectively, our findings identify GLI1 as an important positive regulator of epithelial differentiation, and they offer an explanation for how decreased levels of GLI1 are likely to contribute to the highly metastatic phenotype of PDAC.
Assuntos
Carcinoma Ductal Pancreático/patologia , Transição Epitelial-Mesenquimal , Neoplasias Pancreáticas/patologia , Fatores de Transcrição/antagonistas & inibidores , Junções Aderentes/patologia , Western Blotting , Linhagem Celular Tumoral , Imunofluorescência , Inativação Gênica , Humanos , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
The role of the epithelial-to-mesenchymal transition (EMT) during hepatocellular carcinoma (HCC) progression is well established, however the regulatory mechanisms modulating this phenomenon remain unclear. Here, we demonstrate that transcription factor glioma-associated oncogene 1 (GLI1) modulates EMT through direct up-regulation of SNAI1 and serves as a downstream effector of the transforming growth factor-ß1 (TGFß1) pathway, a well-known regulator of EMT in cancer cells. Overexpression of GLI1 increased proliferation, viability, migration, invasion, and colony formation by HCC cells. Conversely, GLI1 knockdown led to a decrease in all the above-mentioned cancer-associated phenotypes in HCC cells. Further analysis of GLI1 regulated cellular functions showed that this transcription factor is able to induce EMT and identified SNAI1 as a transcriptional target of GLI1 mediating this cellular effect in HCC cells. Moreover, we demonstrated that an intact GLI1-SNAI1 axis is required by TGFß1 to induce EMT in these cells. Together, these findings define a novel cellular mechanism regulated by GLI1, which controls the growth and EMT phenotype in HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Humanos , Microscopia de Fluorescência/métodos , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fenótipo , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição da Família Snail , Transfecção , Proteína GLI1 em Dedos de ZincoRESUMO
TGF-ß Inducible Early Gene-1 (TIEG1) is a Krüppel-like transcription factor (KLF10) that was originally cloned from human osteoblasts as an early response gene to TGF-ß treatment. As reported previously, TIEG1(-/-) mice have decreased cortical bone thickness and vertebral bone volume and have increased spacing between the trabeculae in the femoral head relative to wildtype controls. Here, we have investigated the role of TIEG1 in osteoclasts to further determine their potential role in mediating this phenotype. We have found that TIEG1(-/-) osteoclast precursors differentiated more slowly compared to wildtype precursors in vitro and high RANKL doses are able to overcome this defect. We also discovered that TIEG1(-/-) precursors exhibit defective RANKL-induced phosphorylation and accumulation of NFATc1 and the NFATc1 target gene DC-STAMP. Higher RANKL concentrations reversed defective NFATc1 signaling and restored differentiation. After differentiation, wildtype osteoclasts underwent apoptosis more quickly than TIEG1(-/-) osteoclasts. We observed increased AKT and MEK/ERK signaling pathway activation in TIEG1(-/-) osteoclasts, consistent with the roles of these kinases in promoting osteoclast survival. Adenoviral delivery of TIEG1 (AdTIEG1) to TIEG1(-/-) cells reversed the RANKL-induced NFATc1 signaling defect in TIEG1(-/-) precursors and eliminated the differentiation and apoptosis defects. Suppression of TIEG1 with siRNA in wildtype cells reduced differentiation and NFATc1 activation. Together, these data provide evidence that TIEG1 controls osteoclast differentiation by reducing NFATc1 pathway activation and reduces osteoclast survival by suppressing AKT and MEK/ERK signaling.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Sistema de Sinalização das MAP Quinases , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Osteoclastos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligante RANK/farmacologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1RESUMO
PURPOSE: To determine the concentration of myocilin in primary open-angle glaucoma (POAG) and pseudoexfoliation glaucoma (PEXG) aqueous humor. METHODS: Aqueous humor was collected during surgery from patients with POAG, PEXG, and elective cataract removal (control). Volume-equivalent aqueous samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gels. Quantification of myocilin levels was performed using Western blots probed with 2 independent N-terminal polyclonal anti-myocilin antibodies (AB1 and AB2) followed by densitometry. Myocilin levels in aqueous humor were quantified by plotting the densitometry readings of the aqueous samples against a recombinant myocilin standard curve. Total protein concentration was determined by Bradford protein assay. Transforming growth factor ß 2 levels were assessed by enzyme-linked immunosorbent assay. RESULTS: Myocilin levels are significantly elevated in human POAG aqueous humor when compared with control aqueous humor (AB1: 0.66±0.53 ng/µL vs. 0.23±0.20 ng/µL, P<0.001; AB2: 0.98±0.59 ng/µL vs. 0.65±0.5 ng/µL, P<0.03; mean±SD). Myocilin makes up a larger percent of the total protein in POAG aqueous humor compared with control aqueous (AB1: 0.26±0.20% vs. 0.10±0.20%, P<0.001; AB2: 0.43±0.32% vs. 0.28±0.18%, P<0.05). In contrast to POAG, myocilin levels were not elevated in PEXG aqueous humor when compared with control aqueous humor. No correlation between myocilin and transforming growth factor ß 2 levels was observed. CONCLUSIONS: Myocilin is elevated in POAG, but not in PEXG aqueous humor.
Assuntos
Humor Aquoso/metabolismo , Proteínas do Citoesqueleto/metabolismo , Síndrome de Exfoliação/metabolismo , Proteínas do Olho/metabolismo , Glaucoma de Ângulo Aberto/metabolismo , Glicoproteínas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta2/metabolismoRESUMO
2-Methoxyestradiol (2ME(2)), a physiologic metabolite of 17beta-estradiol (estrogen), has emerged as a promising cancer therapy because of its potent growth-inhibitory and proapoptotic effects on both endothelial and tumor cells. 2ME(2) also suppresses osteoclast differentiation and induces apoptosis of mature osteoclasts, and has been shown to effectively repress bone loss in an animal model of postmenopausal osteoporosis. Given these observations, we have examined whether 2ME(2) could effectively target metastasis to bone, osteolytic tumors, and soft tissue tumors. A 4T1 murine metastatic breast cancer cell line was generated that stably expressed Far Red fluorescence protein (4T1/Red) to visualize tumor development and metastasis to bone. In an intervention study, 4T1/Red cells were injected into bone marrow of the left femur and the mammary pad. In the latter study, 2ME(2) (10, 25, and 50 mg/kg/d) treatment began on the same day as surgery and was continued for the 16-day duration of study. Tumor cell growth and metastasis to bone were monitored and bone volume was determined by micro-computed tomography. 2ME(2) inhibited tumor growth in soft tissue, metastasis to bone, osteolysis, and tumor growth in bone, with maximum effects at 50 mg/kg/d. Furthermore, tumor-induced osteolysis was significantly reduced in mice receiving 2ME(2). In vitro, 2ME(2) repressed osteoclast number by inducing apoptosis of osteoclast precursors as well as mature osteoclasts. Our data support the conclusion that 2ME(2) could be an important new therapy in the arsenal to fight metastatic breast cancer.
Assuntos
Estradiol/análogos & derivados , Neoplasias Mamárias Experimentais/tratamento farmacológico , Osteólise/prevenção & controle , 2-Metoxiestradiol , Animais , Neoplasias Ósseas/prevenção & controle , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Progressão da Doença , Relação Dose-Resposta a Droga , Estradiol/uso terapêutico , Feminino , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Osteoclastos/efeitos dos fármacosRESUMO
The domestic cat (Felix domesticus) is a useful model for understanding the implications of long-term expression and function of normal and mutant myocilin. To better understand the role myocilin has in the cat eye, we isolated and characterized cat myocilin. Oligonucleotides designed against conserved nucleotide regions of myocilin mammalian orthologs were used to PCR amplify a partial cat myocilin cDNA clone. Rapid amplification of cDNA ends (5' and 3' RACE) was used to obtain full-length cat myocilin. The 2125 nucleotide cat myocilin cDNA contains a 490 amino acid open reading frame. Comparison of cat myocilin to human myocilin shows a 87% similarity, including conservation of the N-terminal leucine zipper, N-linked glycosylation site, C-terminal olfactomedin domain, and all five cysteine residues thought to be involved in disulfide bond formation. Expression in a transformed human trabecular cell line or in Crandall feline kidney cells showed cat myocilin was secreted from these cells, similar to human myocilin, suggesting cat myocilin contains a functional signal peptide sequence. In contrast, expression of cat myocilin containing a known human glaucoma-associated mutation (Y423H in cat; Y437H in human) was not secreted. Characterization of cat myocilin will enable long-term studies be performed in Felix domesticus to analyze changes to intraocular pressure and the aqueous outflow pathway following expression of myocilin and glaucoma causing mutations.