RESUMO
The interplay between membrane subregions and receptor tyrosine kinases (RTK) will influence signaling in both normal and pathological RTK conditions. In this study, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor ß (PDGFR-ß) internalizations were investigated by immunofluorescent microscopy following simultaneous treatment with EGF and PDGF-BB. We found that the two receptors utilize separate routes of internalization, which merges in a common perinuclear endosomal compartment after 45 min of stimulation. This is further strengthened when contrasting the recruitment of either EGFR or PDGFR-ß to either clathrin or caveolin-1: PDGFR-ß dissociates from caveolin-1 upon stimulation, and engages clathrin, whilst an increased recruitment of EGFR, to both clathrin and caveolin-1, was observed upon EGF stimulation. The association between EGFR and caveolin-1 is supported by the observation that EGFR was localized in lipid raft associated fractions, whereas PDGFR-ß was not. We also found that disruption of lipid rafts using MßCD led to an increased EGFR dimerization and phosphorylation in response to ligand, as well as a dramatic decrease in AKT- and a smaller but robust decrease in ERK1/2 phosphorylation. This suggest that lipid rafts may be important to effectively connect the EGFR with downstream proteins to facilitate signaling. Our data implies that cholesterol depletion of the plasma membrane affect the signaling of EGFR and PDGFRß differently.
Assuntos
Caveolina 1 , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-akt/metabolismo , Caveolina 1/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Sistema de Sinalização das MAP Quinases , Receptores ErbB/metabolismo , Fosforilação , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Clatrina/metabolismo , Colesterol/metabolismoRESUMO
Interaction of platelet-derived growth factor (PDGF) isoforms with their receptors results in activation and internalization of receptors, with a concomitant activation of downstream signalling pathways. Ubiquitination of PDGFRs serves as a mark to direct the internalization and sorting of the receptors. By overexpressing a panel of deubiquitinating enzymes (DUBs), we found that USP17 and USP4 efficiently deubiquitinate PDGF receptor ß (PDGFRß) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFRß did not affect its stability, but regulated the timing of its trafficking, whereby USP17 prolonged the presence of the receptor at the cell surface, while USP4 affected the speed of trafficking towards early endosomes. Induction of each of the DUBs in BJhTERT fibroblasts and U2OS osteosarcoma cells led to prolonged and/or shifted activation of STAT3 in response to PDGF-BB stimulation, which in turn led to increased transcriptional activity of STAT3. Induction of USP17 promoted acute upregulation of the mRNA expression of STAT3-inducible genes STAT3, CSF1, junB and c-myc, while causing long-term changes in the expression of myc and CDKN1A. Deletion of USP17 was lethal to fibroblasts, while deletion of USP4 led to a decreased proliferative response to stimulation by PDGF-BB. Thus, USP17- and USP4-mediated changes in ubiquitination of PDFGRß lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.
Assuntos
Becaplermina/farmacologia , Endopeptidases/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteases Específicas de Ubiquitina/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Endopeptidases/química , Endopeptidases/genética , Humanos , Mutagênese , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/genética , UbiquitinaçãoRESUMO
Activation of platelet-derived growth factor (PDGF) receptors α and ß (PDGFRα and PDGFRß) at the cell surface by binding of PDGF isoforms leads to internalization of receptors, which affects the amplitude and kinetics of signaling. Ubiquitination of PDGF receptors in response to ligand stimulation is mediated by the Casitas b-lineage lymphoma (Cbl) family of ubiquitin ligases, promoting internalization and serving as a sorting signal for vesicular trafficking of receptors. We report here that another E3 ligase, i.e., tripartite motif-containing protein 21 (TRIM21), contributes to the ubiquitination of PDGFRß in human primary fibroblasts AG1523 and the osteosarcoma cell line U2OS and regulates basal levels of PDGFRß. We found that siRNA-mediated depletion of TRIM21 led to decreased ubiquitination of PDGFRß in response to PDGF-BB stimulation, while internalization from the cell surface and the rate of ligand-induced degradation of the receptor were not affected. Moreover, induction of TRIM21 decreased the levels of PDGFRß in serum-starved cells, and even more in growing cells, in the absence of PDGF stimulation. Consistently, siRNA knockdown of TRIM21 caused accumulation of the total amount of PDGFRß, both in the cytoplasm and on the cell surface, without affecting mRNA levels of the receptor. We conclude that TRIM21 acts post-translationally and maintains basal levels of PDGFRß, thus suggesting that ubiquitination of PDGFRß by TRIM21 may direct a portion of receptor for degradation in growing cells in a ligand-independent manner.
Assuntos
Fator de Crescimento Derivado de Plaquetas , Ubiquitina-Proteína Ligases , Humanos , Proteínas de Transporte/metabolismo , Ligantes , Fosforilação/fisiologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The role of plasma membrane composition and dynamics in the activation process of receptor tyrosine kinases (RTKs) is still poorly understood. In this study we have investigated how signaling via the RTK, platelet-derived growth factor ß-receptor (PDGFR-ß) is affected by Dynasore or Dyngo-4a, which are commonly used dynamin inhibitors. PDGFR-ß preferentially internalizes via clathrin-coated pits and in this pathway, Dynamin II has a major role in the formation and release of vesicles from the plasma membrane by performing the membrane scission. We have found that dynamin inhibitors impedes the activation of PDGFR-ß by impairing ligand-induced dimerization of the receptor monomers, which leads to a subsequent lack of phosphorylation and activation both of receptors and downstream effectors, such as ERK1/2 and AKT. In contrast, dynamin inhibitors did not affect epidermal growth factor receptor (EGFR) dimerization and phosphorylation. Our findings suggest that there is a link between plasma membrane dynamics and PDGFR-ß activation, and that this link is not shared with the epidermal growth factor receptor.
Assuntos
Dinaminas/genética , Multimerização Proteica/efeitos dos fármacos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Dinaminas/antagonistas & inibidores , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Hidrazonas/farmacologia , Ligantes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Naftóis/farmacologia , Fosforilação/efeitos dos fármacos , Multimerização Proteica/genética , Proteínas Proto-Oncogênicas c-akt/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/química , Transdução de Sinais/genéticaRESUMO
Platelet-derived growth factor (PDGF) is a family of growth factors with mitogenic and chemotactic activity. However, uncontrolled and overactivated PDGF signaling has been implicated in a variety of diseases, such as cancers and atherosclerosis. In this context, inhibition of PDGF-PDGFR signaling is of paramount importance in progression of such diseases. The purpose of the current study was to identify novel PDGF-B inhibitors using virtual screening methods. To this end, a combination of molecular modeling techniques such as molecular docking and dynamics simulation, as well as drug likeness filtering criteria, was applied to select anti-PDGF peptidomimetic candidates based on crystallography solved structure of an anti-PDGF-B monoclonal antibody named, MOR8457. In vitro biological assays of the selected compounds revealed two of them being active at micromolar IC50 concentrations. The presented work can provide a framework for systematic peptidomimetic identification for anti-PDGF-B agents from large chemical libraries.
Assuntos
Anticorpos Monoclonais/farmacologia , Descoberta de Drogas , Proteínas Proto-Oncogênicas c-sis/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Anticorpos Monoclonais/química , Células Cultivadas , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Stem cell factor (SCF) is a dimeric molecule that exerts its biological functions by binding to and activating the receptor tyrosine kinase c-Kit. Activation of c-Kit leads to its autophosphorylation and initiation of signal transduction. Signaling proteins are recruited to activated c-Kit by certain interaction domains (e.g., SH2 and PTB) that specifically bind to phosphorylated tyrosine residues in the intracellular region of c-Kit. Activation of c-Kit signaling has been found to mediate cell survival, migration, and proliferation depending on the cell type. Signaling from c-Kit is crucial for normal hematopoiesis, pigmentation, fertility, gut movement, and some aspects of the nervous system. Deregulated c-Kit kinase activity has been found in a number of pathological conditions, including cancer and allergy. The observation that gain-of-function mutations in c-Kit can promote tumor formation and progression has stimulated the development of therapeutics agents targeting this receptor, e.g., the clinically used inhibitor imatinib mesylate. Also other clinically used multiselective kinase inhibitors, for instance, sorafenib and sunitinib, have c-Kit included in their range of targets. Furthermore, loss-of-function mutations in c-Kit have been observed and shown to give rise to a condition called piebaldism. This review provides a summary of our current knowledge regarding structural and functional aspects of c-Kit signaling both under normal and pathological conditions, as well as advances in the development of low-molecular-weight molecules inhibiting c-Kit function.
Assuntos
Proteínas Proto-Oncogênicas c-kit/fisiologia , Transdução de Sinais/fisiologia , Fator de Células-Tronco/fisiologia , Animais , Sobrevivência Celular/fisiologia , Humanos , Fosforilação/fisiologiaRESUMO
Dual specificity phosphatase (DUSP) 4 has been described as a negative regulator of MAP kinase signaling, in particular for the ERK1/2 and JNK pathways. We found that DUSP4 expression was upregulated in response to prolonged platelet-derived growth factor (PDGF)-BB stimulation. The PDGF-BB-induced DUSP4 expression was dependent on ERK1/2, STAT3 and p53. We found that inhibition of ERK1/2 effectively reduced DUSP4 mRNA levels, whereas STAT3 was necessary for maintaining p53 expression. p53 has binding sites in the DUSP4 promoter and was found to promote DUSP4 expression.
Assuntos
Becaplermina/farmacologia , Fosfatases de Especificidade Dupla/genética , Fibroblastos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Regulação para Cima/efeitos dos fármacos , Células Cultivadas , Fosfatases de Especificidade Dupla/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Interferência de RNA , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Protein ubiquitination controls protein stability and subcellular localization of tyrosine kinase receptors, hence affecting signaling both quantitatively and qualitatively. In this report, we demonstrate that, after ligand stimulation, the PDGF ß receptor (PDGFRß) becomes ubiquitinated in a manner requiring both the c-Cbl and Cbl-b ubiquitin ligases. Simultaneous depletion of c-Cbl and Cbl-b resulted in reduced ligand-induced PDGFRß clearance from the cell surface because of reduced endocytosis of the receptor. Cbl-b formed a complex with c-Cbl, as well as with the PDGFRß, in response to PDGF-BB stimulation. We were unable to find a direct interaction between the receptor and c-Cbl, raising the possibility that Cbl-b is necessary for c-Cbl to interact with PDGFRß. Phosphorylated Tyr-1021 in PDGFRß was the primary interaction site for Cbl-b, with some contribution from Tyr-1009. Depletion of c-Cbl and Cbl-b led to an increased ligand-induced tyrosine phosphorylation of the receptor. Several tyrosine residues with elevated phosphorylation (i.e. Tyr-579, Tyr-581, Tyr-1009, and Tyr-1021) have previously been shown to interact with Src kinases and PLCγ. Indeed, in cells depleted of c-Cbl and Cbl-b, both Src and PLCγ phosphorylation were enhanced, whereas activation of other pathways, such as Erk1/2 MAP kinase and Akt, were not affected. In addition, Stat3 phosphorylation, which has been connected to Src activity, was also elevated in cells lacking c-Cbl and Cbl-b. Functionally, we found that cells depleted of c-Cbl and Cbl-b were more prone to migrate toward PDGF-BB, whereas no reproducible effect on cell proliferation could be observed. In conclusion, internalization as well as signaling via PDGFRß are controlled by ubiquitination.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quimiotaxia/fisiologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Becaplermina , Western Blotting , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Tirosina/metabolismo , Ubiquitinação , Quinases da Família src/metabolismoRESUMO
RhoD is a member of the classical Rho GTPases and it has essential roles in the regulation of actin dynamics. RhoD localizes to early endosomes and recycling endosomes, which indicates its important role in the regulation of endosome trafficking. Here, we show that RhoD binds to the Rab5 effector Rabankyrin-5, and RhoD and Rabankyrin-5 colocalize to Rab5-positive endosomes, which suggests a role for Rabankyrin-5 in the coordination of RhoD and Rab5 in endosomal trafficking. Interestingly, depletion of RhoD using siRNA techniques interfered with the internalization of the PDGFß receptor and the subsequent activation of the downstream signaling cascades. Our data suggest that RhoD and Rabankyrin-5 have important roles in coordinating RhoD and Rab activities during internalization and trafficking of activated tyrosine kinase receptors.
Assuntos
Proteínas de Membrana/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Sítios de Ligação , Endossomos/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Ligação a Fosfato , Ligação Proteica , Transporte Proteico , Proteínas rab5 de Ligação ao GTP/química , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genéticaRESUMO
BACKGROUND: The survival of high-grade glioma patients is poor and the treatment of these patients can cause severe side effects. This fosters the necessity to identify prognostic biomarkers, in order to optimize treatment and diminish unnecessary suffering of patients. The aim of this study was to identify prognostic biomarkers for high-grade glioma patients. METHODS: Eleven proteins were selected for analysis due to their suggested importance for survival of patients with other types of cancers and due to a high variation in protein levels between glioma patients (according to the Human Protein Atlas, www.proteinatlas.org). Protein expression patterns of these 11 proteins were analyzed by immunohistochemistry in tumor samples from 97 high-grade glioma patients. The prognostic values of the proteins were analyzed with univariate and multivariate Cox regression analyses for the high-grade glioma patients, including subgroup analyses of histological subtypes and immunohistochemically defined molecular subtypes. RESULTS: The proteins with the most significant (univariate and multivariate p<0.05) correlations were analyzed further with cross-validated Kaplan-Meier analyses for the possibility of predicting survival based on the protein expression pattern of the corresponding candidate. Random Forest classification with variable subset selection was used to analyze if a protein signature consisting of any combination of the 11 proteins could predict survival for the high-grade glioma patients and the subgroup with glioblastoma patients. The proteins which correlated most significantly (univariate and multivariate p<0.05) to survival in the Cox regression analyses were Myc for all high-grade gliomas and FGF2, CA9 and CD44 for the subgroup of proneural gliomas, with FGF2 having a strong negative predictive value for survival. No prognostic signature of the proteins could be found. CONCLUSION: FGF2 is a potential prognostic biomarker for proneural glioma patients, and warrants further investigation.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glioma/metabolismo , Glioma/mortalidade , Proteínas de Neoplasias/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Antígenos de Neoplasias/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Anidrase Carbônica IX , Anidrases Carbônicas/metabolismo , Feminino , Glioma/patologia , Glioma/terapia , Humanos , Receptores de Hialuronatos/metabolismo , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Análise de Regressão , Estudos Retrospectivos , Análise Serial de TecidosRESUMO
Fer is a cytoplasmic tyrosine kinase that is activated in response to platelet-derived growth factor (PDGF) stimulation. In the present report, we show that Fer associates with the activated PDGF ß-receptor (PDGFRß) through multiple autophosphorylation sites, i.e. Tyr-579, Tyr-581, Tyr-740, and Tyr-1021. Using low molecular weight inhibitors, we found that PDGF-BB-induced Fer activation is dependent on PDGFRß kinase activity, but not on the enzymatic activity of Src or Jak kinases. In cells in which Fer was down-regulated using siRNA, PDGF-BB was unable to induce phosphorylation of STAT3, whereas phosphorylations of STAT5, ERK1/2, and Akt were unaffected. PDGF-BB-induced activation of STAT3 occurred also in cells expressing kinase-dead Fer, suggesting a kinase-independent adaptor role of Fer. Expression of Fer was dispensable for PDGF-BB-induced proliferation and migration but essential for colony formation in soft agar. Tumor growth in vivo was delayed in cells depleted of Fer expression. Our data suggest a critical role of Fer in PDGF-BB-induced STAT3 activation and cell transformation.
Assuntos
Indutores da Angiogênese/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias/metabolismo , Proteínas Tirosina Quinases/biossíntese , Proteínas Proto-Oncogênicas c-sis/farmacologia , Fator de Transcrição STAT3/metabolismo , Animais , Becaplermina , Linhagem Celular Tumoral , Movimento Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células NIH 3T3 , Neoplasias/genética , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Tirosina Quinases/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
The prognosis of high-grade glioma patients is poor, and the tumors are characterized by resistance to therapy. The aims of this study were to analyze the prognostic value of the expression of the protein tyrosine phosphatase non-receptor type 6 (PTPN6, also referred to as SHP1) in high-grade glioma patients, the epigenetic regulation of the expression of PTPN6, and the role of its expression in chemotherapy resistance in glioma-derived cells. PTPN6 expression was analyzed with immunohistochemistry in 89 high-grade glioma patients. Correlation between PTPN6 expression and overall survival was analyzed with Kaplan-Meier univariate analysis and Cox regression multivariate analysis. Differences in drug sensitivity to a panel of 16 chemotherapeutic drugs between PTPN6-overexpressing clones and control clones were analyzed in vitro with the fluorometric microculture cytotoxicity assay. Cell cycle analysis was done with Krishan staining and flow cytometry. Apoptosis was analyzed with a cell death detection ELISA kit as well as cleaved caspase-3 and caspase-9 Western blotting. Autophagy was analyzed with LC3B Western blotting. Methylation of the PTPN6 promoter was analyzed with bisulfite pyrosequencing, and demethylation of PTPN6 was done with decitabine treatment. The PTPN6 expression correlated in univariate analysis to poor survival for anaplastic glioma patients (p = 0.026). In glioma-derived cell lines, overexpression of PTPN6 caused increase resistance (p < 0.05) to the chemotherapeutic drugs bortezomib, cisplatin, and melphalan. PTPN6 expression did not affect bortezomib-induced cell cycle arrest, apoptosis, or autophagy. Low PTPN6 promoter methylation correlated to protein expression, and the protein expression was increased upon demethylation in glioma-derived cells. PTPN6 expression may be a factor contributing to poor survival for anaplastic glioma patients, and in glioma-derived cells, its expression is epigenetically regulated and influences the response to chemotherapy.
Assuntos
Neoplasias Encefálicas/mortalidade , Epigênese Genética , Glioma/mortalidade , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Autofagia , Ácidos Borônicos/farmacologia , Bortezomib , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Glioma/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Prognóstico , Regiões Promotoras Genéticas , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Pirazinas/farmacologiaRESUMO
The active form of vitamin D, 1,25-dihydroxyvitamin D3, is known to act via VDR (vitamin D receptor), affecting several physiological processes. In addition, PDIA3 (protein disulphide-isomerase A3) has been associated with some of the functions of 1,25-dihydroxyvitamin D3. In the present study we used siRNA-mediated silencing of PDIA3 in osteosarcoma and prostate carcinoma cell lines to examine the role(s) of PDIA3 for 1,25-dihydroxyvitamin D3-dependent responses. PDIA3 silencing affected VDR target genes and significantly altered the 1,25-dihydroxyvitamin D3-dependent induction of CYP24A1, essential for elimination of excess 1,25-dihydroxyvitamin D3. Also, PDIA3 silencing significantly altered migration and proliferation in prostate PC3 cells, independently of 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 increased thermostability of PDIA3 in cellular thermal shift assay, supporting functional interaction between PDIA3 and 1,25-dihydroxyvitamin D3-dependent pathways. In summary, our data link PDIA3 to 1,25-dihydroxyvitamin D3-mediated signalling, underline and extend its role in proliferation and reveal a novel function in maintenance of 1,25-dihydroxyvitamin D3 levels.
Assuntos
Movimento Celular , Proliferação de Células , Isomerases de Dissulfetos de Proteínas , Receptores de Calcitriol , Vitamina D3 24-Hidroxilase , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Humanos , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/genética , Linhagem Celular Tumoral , Vitamina D3 24-Hidroxilase/genética , Vitamina D3 24-Hidroxilase/metabolismo , Calcitriol/farmacologia , Calcitriol/metabolismo , Inativação Gênica , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/genética , Vitamina D/metabolismo , Vitamina D/farmacologia , Vitamina D/análogos & derivados , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Mammalian target of rapamycin (mTOR) can be found in two multi-protein complexes, i.e. mTORC1 (containing Raptor) and mTORC2 (containing Rictor). Here, we investigated the mechanisms by which mTORC1 and mTORC2 are activated and their downstream targets in response to platelet-derived growth factor (PDGF)-BB treatment. Inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited PDGF-BB activation of both mTORC1 and mTORC2. We found that in Rictor-null mouse embryonic fibroblasts, or after prolonged rapamycin treatment of NIH3T3 cells, PDGF-BB was not able to promote phosphorylation of Ser473 in the serine/threonine kinase Akt, whereas Thr308 phosphorylation was less affected, suggesting that Ser473 in Akt is phosphorylated in an mTORC2-dependent manner. This reduction in Akt phosphorylation did not influence the phosphorylation of the S6 protein, a well established protein downstream of mTORC1. Consistently, triciribine, an inhibitor of the Akt pathway, suppressed PDGF-BB-induced Akt phosphorylation without having any effect on S6 phosphorylation. Thus, mTORC2 does not appear to be upstream of mTORC1. We could also demonstrate that in Rictor-null cells the phosphorylation of phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC) was impaired, and the PKCα protein levels strongly reduced. Furthermore, interfering with the PLCγ/Ca2+/PKC pathway inhibited PDGF-BB-induced Akt phosphorylation. In addition, PDGF-BB-induced activation of mTORC1, as measured by phosphorylation of the downstream S6 protein, was dependent on phospholipase D (PLD). It has been shown that Erk1/2 MAP-kinase directly phosphorylates and activates mTORC1; in partial agreement with this finding, we found that a Mek1/2 inhibitor delayed S6 phosphorylation in response to PDGF-BB, but it did not block it. Thus, whereas both mTORC1 and mTORC2 are activated in a PI3K-dependent manner, different additional signaling pathways are needed. mTORC1 is activated in a PLD-dependent manner and promotes phosphorylation of the S6 protein, whereas mTORC2, in concert with PLCγ signaling, promotes Akt phosphorylation.
RESUMO
The importance of identifying VEGF-independent pathways in pathological angiogenesis is increasingly recognized as a result of the emerging drug resistance to anti-VEGF therapies. PDGF-CC is the third member of the PDGF family discovered after more than two decades of studies on PDGF-AA and PDGF-BB. The biological function of PDGF-CC and the underlying cellular and molecular mechanisms remain largely unexplored. Here, using different animal models, we report that PDGF-CC inhibition by neutralizing antibody, shRNA, or genetic deletion suppressed both choroidal and retinal neovascularization. Importantly, we revealed that PDGF-CC targeting acted not only on multiple cell types important for pathological angiogenesis, such as vascular mural and endothelial cells, macrophages, choroidal fibroblasts and retinal pigment epithelial cells, but also on the expression of other important angiogenic genes, such as PDGF-BB and PDGF receptors. At a molecular level, we found that PDGF-CC regulated glycogen synthase kinase (GSK)-3beta phosphorylation and expression both in vitro and in vivo. Activation of GSK3beta impaired PDGF-CC-induced angiogenesis, and inhibition of GSK3beta abolished the antiangiogenic effect of PDGF-CC blockade. Thus, we identified PDGF-CC as an important candidate target gene for antiangiogenic therapy, and PDGF-CC inhibition may be of therapeutic value in treating neovascular diseases.
Assuntos
Linfocinas/genética , Neovascularização Patológica/genética , Fator de Crescimento Derivado de Plaquetas/genética , Interferência de RNA , Animais , Anticorpos Neutralizantes/farmacologia , Becaplermina , Western Blotting , Células Cultivadas , Embrião de Galinha , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/prevenção & controle , Feminino , Imunofluorescência , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Linfocinas/imunologia , Linfocinas/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Fosforilação , Fator de Crescimento Derivado de Plaquetas/imunologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-sis , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: The platelet-derived growth factor (PDGF) family of ligands exerts their cellular effects by binding to α- and ß-tyrosine kinase receptors (PDGFRα and PDGFRß, respectively). SUMOylation is an important posttranslational modification (PTM) which regulates protein stability, localization, activation and protein interactions. A mass spectrometry screen has demonstrated SUMOylation of PDGFRα. However, the functional role of SUMOylation of PDGFRα has remained unknown. RESULTS: In the present study, we validated that PDGFRα is SUMOylated on lysine residue 917 as was previously reported using a mass spectrometry approach. Mutation of lysine residue 917 to arginine (K917R) in PDGFRα substantially decreased SUMOylation, indicating that this amino acid residue is a major SUMOylation site. Whereas no difference in the stability of wild-type and mutant receptor was observed, the K917R mutant PDGFRα was less ubiquitinated than wild-type PDGFRα. The internalization and trafficking of the receptor to early and late endosomes were not affected by the mutation, neither was the localization of the PDGFRα to Golgi. However, the K917R mutant PDGFRα showed delayed activation of PLC-γ and enhanced activation of STAT3. Functional assays showed that the mutation of K917 of PDGFRα decreased cell proliferation in response to PDGF-BB stimulation. CONCLUSIONS: SUMOylation of PDGFRα decreases ubiquitination of the receptor and affects ligand-induced signaling and cell proliferation.
Assuntos
Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Sumoilação , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fosfolipase C gama/metabolismo , Lisina/metabolismo , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo , Proliferação de Células , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
Protein-tyrosine phosphatases (PTPs) are important regulators of cellular signaling and changes in PTP activity can contribute to cell transformation. Little is known about the role of PTPs in Acute Myeloid Leukemia (AML). The aim of this study was therefore to establish a PTP expression profile in AML cells and to explore the possible role of FLT3 ITD (Fms-like tyrosine kinase 3 with internal tandem duplication), an important oncoprotein in AML for PTP gene expression. PTP mRNA expression was analyzed in AML cells from patients and in cell lines using a RT-qPCR platform for detection of transcripts of 92 PTP genes. PTP mRNA expression was also analyzed based on a public microarray data set for AML patients. Highly expressed PTPs in AML belong to all PTP subfamilies. Very abundantly expressed PTP genes include PTPRC, PTPN2, PTPN6, PTPN22, DUSP1, DUSP6, DUSP10, PTP4A1, PTP4A2, PTEN, and ACP1. PTP expression was further correlated with the presence of FLT3 ITD, focusing on a set of highly expressed dual-specificity phosphatases (DUSPs). Elevated expression of DUSP6 in patients harboring FLT3 ITD was detected in this analysis. The mechanism and functional role of FLT3 ITD-mediated upregulation of DUSP6 was then explored using pharmacological inhibitors of FLT3 ITD signal transduction and si/shRNA technology in human and murine cell lines. High DUSP6 expression was causally associated with the presence of FLT3 ITD and dependent on FLT3 ITD kinase activity and ERK signaling. DUSP6 depletion moderately increased ERK1/2 activity but attenuated FLT3 ITD-dependent cell proliferation of 32D cells. In conclusion, DUSP6 may play a contributing role to FLT3 ITD-mediated cell transformation.
RESUMO
BACKGROUND: Leucine-rich and immunoglobulin-like domains 1-3 (LRIG1-3) proteins have been implicated in the regulation of EGFR signalling. In the present study, we investigated the clinical implications of the expression of EGFR and LRIG1-3 in oesophageal carcinoma, as well as the correlation between their expression levels and the chemosensitivity of oesophageal carcinoma cell lines. PATIENTS AND METHODS: Tumours from 80 patients with oesophageal carcinoma were investigated for the expression of EGFR and LRIG proteins by immunohistochemistry. Oesophageal carcinoma cell lines were investigated for their expression of EGFR and LRIG1, 2, and 3 by quantitative real time RT-PCR and for their sensitivity to commonly used chemotherapeutics by a cytotoxicity assay. RESULTS AND DISCUSSION: Based on a total score of intensity and expression rates, a trend towards survival difference was found for EGFR (p = 0.09) and LRIG2 (p = 0.18) whereas for LRIG1 and -3 there was no trend towards any association with survival. Correlation analysis revealed a correlation with the clinical expression of EGFR and LRIG3 (p = 0.0007). Significant correlations were found between LRIG1 expression levels and sensitivity to cisplatin (r = -0.74), docetaxel (r = -0.69), and vinorelbine (r = -0.82) in oesophageal carcinoma cell lines. EGFR and the LRIG proteins may be functionally involved in oesophageal carcinoma, but larger materials are needed to fully elucidate the clinical implication.
Assuntos
Carcinoma/metabolismo , Receptores ErbB/metabolismo , Neoplasias Esofágicas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Idoso , Antineoplásicos/uso terapêutico , Carcinoma/tratamento farmacológico , Carcinoma/mortalidade , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Docetaxel , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/mortalidade , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Suécia/epidemiologia , Taxoides/uso terapêutico , Resultado do Tratamento , Vimblastina/análogos & derivados , Vimblastina/uso terapêutico , VinorelbinaRESUMO
VEGF-B, a homolog of VEGF discovered a long time ago, has not been considered an important target in antiangiogenic therapy. Instead, it has received little attention from the field. In this study, using different animal models and multiple types of vascular cells, we revealed that although VEGF-B is dispensable for blood vessel growth, it is critical for their survival. Importantly, the survival effect of VEGF-B is not only on vascular endothelial cells, but also on pericytes, smooth muscle cells, and vascular stem/progenitor cells. In vivo, VEGF-B targeting inhibited both choroidal and retinal neovascularization. Mechanistically, we found that the vascular survival effect of VEGF-B is achieved by regulating the expression of many vascular prosurvival genes via both NP-1 and VEGFR-1. Our work thus indicates that the function of VEGF-B in the vascular system is to act as a "survival," rather than an "angiogenic" factor and that VEGF-B inhibition may offer new therapeutic opportunities to treat neovascular diseases.
Assuntos
Neovascularização Patológica , Fator B de Crescimento do Endotélio Vascular/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Genoma , Membro Posterior/irrigação sanguínea , Isquemia/genética , Isquemia/metabolismo , Camundongos , Camundongos Knockout , Ratos , Retina/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Regulação para Cima , Fator B de Crescimento do Endotélio Vascular/deficiência , Fator B de Crescimento do Endotélio Vascular/genéticaRESUMO
It has become clear that lipid rafts functions as signaling hotspots connecting cell surface receptors to intracellular signaling pathways. However, the exact involvement of lipid rafts in receptor tyrosine kinase signaling is still poorly understood. In this study, we have analyzed platelet-derived growth factor (PDGF) receptor ß (PDGFR-ß) signaling in two different cell lines depleted of cholesterol, and as a consequence, disruption of lipid rafts. Cholesterol depletion of BJ-hTERT fibroblasts using methyl-ß-cyclodextrin (MßCD) did not affect PDGFR-ß activation as measured by its tyrosine phosphorylation. However, we did observe a small reduction in AKT phosphorylation and a more robust decrease of ERK1/2 activation. In contrast, in the osteosarcoma cell line U2OS, we noticed a deficient receptor activation. Interestingly, in U2OS cells, the ERK1/2 pathway was unaffected, but instead AKT and SRC signaling was reduced. These results suggest that cell type specific wiring of signaling pathways can lead to differential sensitivity to cholesterol depletion. Furthermore, MßCD treatment had a much more pronounced morphological effect on U2OS compared to BJ-hTERT cells. This is consistent with a previous report claiming that cancer cells are more sensitive to cholesterol depletion than normal cells. Our data supports the possibility that cholesterol lowering drugs may impede tumor growth.