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1.
Oncogene ; 38(32): 6065-6081, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31273338

RESUMO

Long non-coding RNA (lncRNA) plays an important role in malignant tumor occurrence, development, and chemoresistance, but the mechanism of how they affect nasopharyngeal cancer (NPC) paclitaxel chemosensitivity is unclear. In this study, lncRNA array of CNE-1 and HNE-2 paclitaxel-resistant cells and their parental strains revealed that the paclitaxel-resistant strains had significantly lower MRVI1-AS1 (murine retrovirus integration site 1 homolog antisense RNA 1) expression than the parental strains, and that MRVI1-AS1 overexpression in vitro and in vivo increased paclitaxel chemosensitivity. Further, MRVI1-AS1 upregulated ATF3 (activating transcription factor 3) by simultaneously inhibiting miR-513a-5p (microRNA-513a-5p) and miR-27b-3p expression levels to increase NPC paclitaxel chemosensitivity. Chromatin immunoprecipitation and quantitative real-time PCR showed that ATF3 could feed-back MRVI1-AS1 regulation positively. Furthermore, MRVI1-AS1 and ATF3 could form a positive feedback loop, which promoted the expression of RASSF1 (Ras association domain family member 1), a Hippo-TAZ (tafazzin) signaling pathway regulatory factor, thereby inhibiting TAZ expression. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay and flow cytometry showed that the decreased TAZ increased NPC cell paclitaxel chemosensitivity. Overall, the results indicate that the MRVI1-AS1/ATF3 signaling pathway can increase NPC paclitaxel chemosensitivity by modulating the Hippo-TAZ signaling pathway. Therefore, targeting the loop may be a new NPC treatment strategy.


Assuntos
Fator 3 Ativador da Transcrição/genética , Proteínas de Membrana/genética , Carcinoma Nasofaríngeo/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico , Paclitaxel/uso terapêutico , Fosfoproteínas/genética , RNA Antissenso/fisiologia , Células A549 , Fator 3 Ativador da Transcrição/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Células MCF-7 , Proteínas de Membrana/antagonistas & inibidores , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Fosfoproteínas/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Gene ; 712: 143956, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31271843

RESUMO

Gastric cancer represents a common malignancy of digestive tract with high incidence and mortality. Increasing evidence suggests that the growth of gastric tumor cells relies largely on aerobic glycolysis. Currently, many potential anti-cancer candidates are derived from natural products. Here, we evaluated the effects of oleanolic acid (OA), a triterpenoid component widely found in the plants of Oleaceae family, on aerobic glycolysis and proliferation in human MKN-45 and SGC-7901 gastric cancer cells. Our results demonstrated that OA reduced the viability and proliferation of gastric cancer cells and inhibited the expression of cyclin A and cyclin-dependent kinase 2. OA blocked glycolysis in these cells evidenced by decreases in the uptake and consumption of glucose, intracellular lactate levels and extracellular acidification rate. Glycolysis inhibitor 2-deoxy-d-glucose, similar to OA, suppressed gastric cancer cell proliferation. OA also decreased the expression and intracellular activities of glycolysis rate-limiting enzymes hexokinase 2 (HK2) and phosphofructokinase 1 (PFK1). Moreover, OA downregulated the expression of hypoxia inducible factor-1α (HIF-1α) and decreased its nuclear abundance. Upregulation of HIF-1α by deferoxamine rescued OA-inhibited HK2 and PFK1. Furthermore, OA reduced the nuclear abundance of yes-associated protein (YAP) in gastric tumor cells. YAP inhibitor verteporfin, similar to OA, downregulated the expression of HIF-1α and glycolytic enzymes in gastric cancer cells; whereas overexpression of YAP abrogated all these effects of OA. Collectively, inhibition of YAP was responsible for OA blockade of HIF-1α-mediated aerobic glycolysis and proliferation in human gastric tumor cells. OA could be developed as a promising candidate for gastric cancer treatment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ácido Oleanólico/farmacologia , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Neoplasias Gástricas/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Ciclo do Ácido Cítrico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise , Humanos , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Estômago/patologia , Neoplasias Gástricas/tratamento farmacológico , Fatores de Transcrição
3.
Future Oncol ; 15(13): 1535-1543, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31066301

RESUMO

YAP, acting as a crucial transcription factor in nucleus, regulates the organ size, tissue homeostasis and tumorigenesis. Dysregulation of Hippo-YAP pathway brings a significant impact on the occurrence and development of various tumor types. Moreover, regulation of YAP/TAZ far exceeds the core kinase of the Hippo pathway, and gradually opens up new therapeutic targets. For the moment, chemotherapy together with radiotherapy act as routine methods to prolong the lives of cancer patients. Seeking more effective anti-neoplastic agents seems to be the urgent problem. This brief review focuses on the research progress of YAP inhibitors as the antineoplastic targets. Small molecule inhibitors or drugs have been discovered including verteporfin, dasatinib, statins, A35, JQ1, norcantharidin, agave, MLN8237, dobutamine and peptide-based YAP inhibitors. We are trying to seek novel therapies from the relationship between known drugs and potential mechanisms.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Fosfoproteínas/antagonistas & inibidores , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Prognóstico , Fatores de Transcrição
4.
Int J Mol Sci ; 20(10)2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31100813

RESUMO

The L-type calcium channel blocker fendiline has been shown to interfere with Ras-dependent signaling in K-Ras mutant cancer cells. Earlier studies from our lab had shown that treatment of pancreatic cancer cells with fendiline causes significant cytotoxicity and interferes with proliferation, survival, migration, invasion and anchorage independent growth. Currently there are no effective therapies to manage PDACs. As fendiline has been approved for treatment of patients with angina, we hypothesized that, if proven effective, combinatorial therapies using this agent would be easily translatable to clinic for testing in PDAC patients. Here we tested combinations of fendiline with gemcitabine, visudyne (a YAP1 inhibitor) or tivantinib (ARQ197, a c-Met inhibitor) for their effectiveness in overcoming growth and oncogenic characteristics of PDAC cells. The Hippo pathway component YAP1 has been shown to bypass K-Ras addiction, and allow tumor growth, in a Ras-null mouse model. Similarly, c-Met expression has been associated with poor prognosis and metastasis in PDAC patients. Our results presented here show that combinations of fendiline with these inhibitors show enhanced anti-tumor activity in Panc1, MiaPaCa2 and CD18/HPAF PDAC cells, as evident from the reduced viability, migration, anchorage-independent growth and self-renewal. Biochemical analysis shows that these agents interfere with various signaling cascades such as the activation of Akt and ERK, as well as the expression of c-Myc and CD44 that are altered in PDACs. These results imply that inclusion of fendiline may improve the efficacy of various chemotherapeutic agents that could potentially benefit PDAC patients.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Desoxicitidina/análogos & derivados , Fendilina/farmacologia , Pirrolidinonas/farmacologia , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Verteporfina/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinógenos , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Humanos , Concentração Inibidora 50 , Camundongos , Metástase Neoplásica , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Fosfoproteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores
5.
World J Gastroenterol ; 25(15): 1797-1816, 2019 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-31057295

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) remains a deadly disease with no efficacious treatment options. PDAC incidence is projected to increase, which may be caused at least partially by the obesity epidemic. Significantly enhanced efforts to prevent or intercept this cancer are clearly warranted. Oncogenic KRAS mutations are recognized initiating events in PDAC development, however, they are not entirely sufficient for the development of fully invasive PDAC. Additional genetic alterations and/or environmental, nutritional, and metabolic signals, as present in obesity, type-2 diabetes mellitus, and inflammation, are required for full PDAC formation. We hypothesize that oncogenic KRAS increases the intensity and duration of the growth-promoting signaling network. Recent exciting studies from different laboratories indicate that the activity of the transcriptional co-activators Yes-associated protein (YAP) and WW-domain-containing transcriptional co-activator with PDZ-binding motif (TAZ) play a critical role in the promotion and maintenance of PDAC operating as key downstream target of KRAS signaling. While initially thought to be primarily an effector of the tumor-suppressive Hippo pathway, more recent studies revealed that YAP/TAZ subcellular localization and co-transcriptional activity is regulated by multiple upstream signals. Overall, YAP has emerged as a central node of transcriptional convergence in growth-promoting signaling in PDAC cells. Indeed, YAP expression is an independent unfavorable prognostic marker for overall survival of PDAC. In what follows, we will review studies implicating YAP/TAZ in pancreatic cancer development and consider different approaches to target these transcriptional regulators.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pancreáticas/genética , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Reposicionamento de Medicamentos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Metformina/farmacologia , Metformina/uso terapêutico , Terapia de Alvo Molecular/métodos , Mutação , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Fosfoproteínas/antagonistas & inibidores , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/genética
6.
Nat Commun ; 10(1): 1464, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30931946

RESUMO

Stem cell-derived insulin-producing beta cells (SC-ß) offer an inexhaustible supply of functional ß cells for cell replacement therapies and disease modeling for diabetes. While successful directed differentiation protocols for this cell type have been described, the mechanisms controlling its differentiation and function are not fully understood. Here we report that the Hippo pathway controls the proliferation and specification of pancreatic progenitors into the endocrine lineage. Downregulation of YAP, an effector of the pathway, enhances endocrine progenitor differentiation and the generation of SC-ß cells with improved insulin secretion. A chemical inhibitor of YAP acts as an inducer of endocrine differentiation and reduces the presence of proliferative progenitor cells. Conversely, sustained activation of YAP results in impaired differentiation, blunted glucose-stimulated insulin secretion, and increased proliferation of SC-ß cells. Together these results support a role for YAP in controlling the self-renewal and differentiation balance of pancreatic progenitors and limiting endocrine differentiation in vitro.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Diferenciação Celular/genética , Secreção de Insulina/genética , Células Secretoras de Insulina/citologia , Fosfoproteínas/genética , Células-Tronco Pluripotentes/citologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Regulação para Baixo , Células HEK293 , Humanos , Imuno-Histoquímica , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Fosfoproteínas/antagonistas & inibidores , Células-Tronco Pluripotentes/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição
7.
Artif Cells Nanomed Biotechnol ; 47(1): 1273-1280, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30963795

RESUMO

Pladienolide B is a potent cancer cell growth inhibitor that targets the SF3b1 subunit of the spliceosome. There is considerable interest in the compound as a tool to study SF3b1 function in cancer. However, so far little information is available on the molecular mechanism of SF3b1 eliciting apoptosis in cancer cells. Here, we investigated the molecular mechanism of SF3b1 eliciting apoptosis in human cervical carcinoma cells. We demonstrated that inhibition of SF3b1 by pladienolide B inhibited proliferation of HeLa cells at low nanomolar concentrations in a dose- and time-dependent manner. It also induced G2/M phase arrest and significant rise of apoptotic cells. Moreover, it is indicated that inhibition of SF3b1 by pladienolide B induced Tap73/ΔNp73 expression and consequently down-regulated Bax/Bcl-2 ratio, cytochrome c release and caspase-3 expression. Thus, our results showed that SF3b1 plays a pivotal role in cycle arrest, apoptosis induction, and p73 splicing in human cervical carcinoma cells, suggesting that SF3b1 could be used as a potential candidate for cervical cancer therapy.


Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Macrolídeos/farmacologia , Fosfoproteínas/antagonistas & inibidores , Fatores de Processamento de RNA/antagonistas & inibidores , Processamento de RNA/efeitos dos fármacos , Proteína Tumoral p73/genética , Neoplasias do Colo do Útero/patologia , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia
8.
Molecules ; 24(3)2019 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-30754629

RESUMO

Treatment of colorectal cancer mostly relies on traditional therapeutic approaches, such as surgery and chemotherapy. Limited options of targeted therapy for colorectal cancer narrowly focus on blocking cancer-generic targets VEGFR and EGFR. Identifying the oncogenic drivers, understanding their contribution to proliferation, and finding inhibitors to block such drivers are the keys to developing targeted therapy for colorectal cancer. In this study, ten colorectal cancer cell lines were screened against a panel of protein kinase inhibitors blocking key oncogenic signaling pathways. The results show that four of the 10 cell lines did not respond to any kinase inhibitors significantly, the other six were mildly inhibited by AZD-6244, BMS-754807, and/or dasatinib. Mechanistic analyses demonstrate that these inhibitors independently block the MAP kinase pathway, IR/IGF-1R/AKT pathway, and Src kinases, suggesting a multi-driver nature of proliferative signaling in these cells. Most of these cell lines were potently and synergistically inhibited by pair-wise combinations of these drugs. Furthermore, seven of the 10 cell lines were inhibited by the triple combination of AZD-6244/BMS-754807/dasatinib with IC50's between 10 and 84 nM. These results suggest that combination targeted therapy may be an effective strategy against colorectal cancer.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzimidazóis/farmacologia , Neoplasias Colorretais/metabolismo , Dasatinibe/farmacologia , Redes Reguladoras de Genes/efeitos dos fármacos , Pirazóis/farmacologia , Triazinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Células HCT116 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Terapia de Alvo Molecular , Fosfoproteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo , Transdução de Sinais/efeitos dos fármacos
9.
Oncol Rep ; 41(3): 1455-1475, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30628704

RESUMO

The Hippo signaling pathway is considered to be a tissue growth regulator and tumor suppressor pathway that controls cell proliferation, differentiation, survival, regeneration and tissue homeostasis. Defects in Hippo kinases and hyperactivation of transcriptional co­activator with PDZ­binding motif and Yes­associated protein (YAP) may contribute to the development of different types of cancer. The Hippo pathway is regulated in a variety of way, of which ubiquitination is of considerable importance. Ubiquitination is a crucial post­translational protein modification in cancer cells and is an applicable target for pharmacological intervention. Ubiquitin modifications are involved in regulating various physiological processes and are counteracted by deubiquitination. Imbalanced ubiquitination­deubiquitination is closely associated with tumor initiation and progression. Therefore, the examination of the specific association between the Hippo pathway and ubiquitination is of interest. The present study reviews the modulatory mechanism of ubiquitination­deubiquitination in the Hippo signaling pathway, the recent progress in identifying therapeutic targets and strategies, and the future directions in the field that may contribute to better tumor diagnosis and treatment.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Enzimas Desubiquitinantes/metabolismo , Progressão da Doença , Humanos , Neoplasias/tratamento farmacológico , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Fatores de Transcrição , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo
10.
Oncogene ; 38(11): 1832-1844, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30356139

RESUMO

Pancreatic cancer remains one of the deadliest human cancers despite current advances in conventional therapeutics including surgery and adjuvant therapies. Here, we showed that LZ1, a peptide derived from a snake venom cathelicidin, significantly inhibited growth of pancreatic cancer cells by inducing autophagy-dependent cell death both in vitro and in vivo. The LZ1-induced cell death was blocked by pharmacological or genetic inhibition of autophagy. In orthotopic model of pancreatic cancer, systemic administration of LZ1 (1-4 mg/kg) exhibited remarkable antitumor efficacy, significantly prolonged mice survival, and showed negligible adverse effects by comparison with gemcitabine (20 mg/kg). Mechanistic studies revealed that LZ1 acts through binding to nucleolin, whose expression on cell surface is frequently increased in pancreatic cancer cells. LZ1 binding triggers degradation of surface-expressed nucleolin. This leads to activation of 5'-AMP kinase which results in suppression of mTORC1 activity and induction of autophagic flux. These data suggest that LZ1, targeting nucleolin-AMPK-autophagy axis, is a promising lead for the development of therapeutic agents against pancreatic cancer.


Assuntos
Adenilato Quinase/metabolismo , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Fragmentos de Peptídeos/uso terapêutico , Fosfoproteínas/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Animais , Antígenos de Superfície/efeitos dos fármacos , Antígenos de Superfície/metabolismo , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Morte Celular/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular/métodos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fragmentos de Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cell Mol Life Sci ; 76(2): 381-395, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30368549

RESUMO

Sister chromatid cohesion plays a key role in ensuring precise chromosome segregation during mitosis, which is mediated by the multisubunit cohesin complex. However, the molecular regulation of cohesin subunits stability remains unclear. Here, we show that NudCL2 (NudC-like protein 2) is essential for the stability of cohesin subunits by regulating Hsp90 ATPase activity in mammalian cells. Depletion of NudCL2 induces mitotic defects and premature sister chromatid separation and destabilizes cohesin subunits that interact with NudCL2. Similar defects are also observed upon inhibition of Hsp90 ATPase activity. Interestingly, ectopic expression of Hsp90 efficiently rescues the protein instability and functional deficiency of cohesin induced by NudCL2 depletion, but not vice versa. Moreover, NudCL2 not only binds to Hsp90, but also significantly modulates Hsp90 ATPase activity and promotes the chaperone function of Hsp90. Taken together, these data suggest that NudCL2 is a previously undescribed Hsp90 cochaperone to modulate sister chromatid cohesion by stabilizing cohesin subunits, providing a hitherto unrecognized mechanism that is crucial for faithful chromosome segregation during mitosis.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas de Ligação a DNA , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Células HeLa , Humanos , Microscopia de Fluorescência , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estabilidade Proteica , Subunidades Proteicas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Imagem com Lapso de Tempo
12.
ChemMedChem ; 14(1): 119-131, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30548204

RESUMO

There is growing interest in the use of structure-based virtual screening to identify small molecules that inhibit challenging protein-protein interactions (PPIs). In this study, we investigated how effectively chemical library members docked at the PPI interface mimic the position of critical side-chain residues known as "hot spots". Three compound collections were considered, a commercially available screening collection (ChemDiv), a collection of diversity-oriented synthesis (DOS) compounds that contains natural-product-like small molecules, and a library constructed using established reactions (the "screenable chemical universe based on intuitive data organization", SCUBIDOO). Three different tight PPIs for which hot-spot residues have been identified were selected for analysis: uPAR⋅uPA, TEAD4⋅Yap1, and CaV α⋅CaV ß. Analysis of library physicochemical properties was followed by docking to the PPI receptors. A pharmacophore method was used to measure overlap between small-molecule substituents and hot-spot side chains. Fragment-like conformationally restricted small molecules showed better hot-spot overlap for interfaces with well-defined pockets such as uPAR⋅uPA, whereas better overlap was observed for more complex DOS compounds in interfaces lacking a well-defined binding site such as TEAD4⋅Yap1. Virtual screening of conformationally restricted compounds targeting uPAR⋅uPA and TEAD4⋅Yap1 followed by experimental validation reinforce these findings, as the best hits were fragment-like and had few rotatable bonds for the former, while no hits were identified for the latter. Overall, such studies provide a framework for understanding PPIs in the context of additional chemical matter and new PPI definitions.


Assuntos
Produtos Biológicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Produtos Biológicos/síntese química , Produtos Biológicos/química , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
13.
Oncogene ; 38(1): 120-139, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30082911

RESUMO

Glioblastomas (GBM) are the most aggressive brain cancers without effective therapeutics. The Hippo pathway transcriptional coactivators YAP/TAZ were implicated as drivers in GBM progression and could be therapeutic targets. Here we found in an unbiased screen of 1650 compounds that amlodipine is able to inhibit survival of GBM cells by suppressing YAP/TAZ activities. Instead of its known function as an L-type calcium channel blocker, we found that amlodipine is able to activate Ca2+ entry by enhancing store-operated Ca2+ entry (SOCE). Amlodipine as well as approaches that cause store depletion and activate SOCE trigger phosphorylation and activation of Lats1/2, which in turn phosphorylate YAP/TAZ and prevent their accumulation in the cell nucleus. Furthermore, we identified that protein kinase C (PKC) beta II is a major mediator of Ca2+-induced Lats1/2 activation. Ca2+ induces accumulation of PKC beta II in an actin cytoskeletal compartment. Such translocation depends on inverted formin-2 (INF2). Depletion of INF2 disrupts both PKC beta II translocation and Lats1/2 activation. Functionally, we found that elevation of cytosolic Ca2+ or PKC beta II expression inhibits YAP/TAZ-mediated gene transcription. In vivo PKC beta II expression inhibits GBM tumor growth and prolongs mouse survival through inhibition of YAP/TAZ in an orthotopic mouse xenograft model. Our studies indicate that Ca2+ is a crucial intracellular cue that regulates the Hippo pathway and that triggering SOCE could be a strategy to target YAP/TAZ in GBM.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Anlodipino/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Glioblastoma/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Fosfoproteínas/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Ionomicina/farmacologia , Camundongos , Camundongos Nus , Proteínas de Neoplasias/fisiologia , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genética , Proteína ORAI1/fisiologia , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Proteína Quinase C beta/fisiologia , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Tapsigargina/farmacologia , Fatores de Transcrição/genética
14.
Stem Cell Reports ; 11(6): 1357-1364, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30449705

RESUMO

Specifying the primitive streak (PS) guides stem cell differentiation in vitro; however, much remains to be learned about the transcription networks that direct anterior and posterior PS cells (APS and PPS, respectively) to differentiate to distinct mesendodermal subpopulations. Here, we show that APS genes are predominantly induced in YAP1-/- human embryonic stem cells (hESCs) in response to ACTIVIN. This finding establishes the Hippo effector YAP1 as a master regulator of PS specification, functioning to repress ACTIVIN-regulated APS genes in hESCs. Moreover, transient exposure of wild-type hESCs to dasatinib, a potent C-SRC/YAP1 inhibitor, enables differentiation to APS-derived endoderm and cardiac mesoderm in response to ACTIVIN. Importantly, these cells can differentiate efficiently to normal beating cardiomyocytes without the cytoskeletal defect seen in YAP1-/- hESC-derived cardiomyocytes. Overall, we uncovered an induction mechanism to generate APS cells using a cocktail of ACTIVIN and YAP1i molecules that holds practical implications for hESC and induced pluripotent stem cell differentiation into distinct mesendodermal lineages.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/metabolismo , Ativinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Dasatinibe/farmacologia , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosfoproteínas/antagonistas & inibidores , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Fatores de Transcrição
15.
Cell Physiol Biochem ; 51(2): 842-853, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30466081

RESUMO

BACKGROUND/AIMS: Increasing wall stress or biomechanical stretch experienced by arteries influences the initiation of atherosclerotic lesions. This initiation is mediated by Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), which are both effectors of the Hippo pathway. In this study, the functional roles of YAP/TAZ proteins in the regulation of the stretch-mediated programing of human umbilical arterial smooth muscle cells (HUASMCs) to a proliferative phenotype were examined. METHODS: HUASMCs were seeded on a Matrigel-coated silicone chamber and subjected to biomechanical stretch for 24 h after 48 h of growth. YAP/TAZ small interfering RNA was used to specifically knockdown YAP/ TAZ expression in HUASMCs. RESULTS: We observed that YAP/TAZ activation via biomechanical stretching is involved in the regulation of critical aspects of the HUASMC phenotypic switch. YAP/TAZ knockdown significantly attenuated the stretch-induced proliferative and pro-inflammatory phenotypes in HUASMCs. Furthermore, treatment with atorvastatin, an anti-atherosclerotic drug, attenuated the stretch-induced phenotypic switch of HUASMCs from the contractile to synthetic state by suppressing YAP/TAZ expression. Additional investigations demonstrated the role of stretch in inhibiting the Hippo pathway, leading to the activation of PI3-kinase (PI3K) and phosphoinositide dependent kinase (PDK1); the key molecule for the regulation of the PDK1 and Hippo complex interaction was Sav1. These results showed the importance of YAP/TAZ activation, induced by biomechanical stretch, in promoting atheroprone phenotypes in HUASMCs. CONCLUSION: Taken together, our findings revealed a mechanism by which YAP/TAZ activation contributes to the pathogenesis of atherosclerosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Remodelação Vascular , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Atorvastatina/farmacologia , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Proliferação de Células , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Mecânico , Fatores de Transcrição/antagonistas & inibidores , Artérias Umbilicais/citologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
16.
Braz J Med Biol Res ; 52(1): e7914, 2018 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-30484494

RESUMO

Yes-associated protein (YAP) is an important regulator of cellular proliferation and transdifferentiation. However, little is known about the mechanisms underlying myofibroblast transdifferentiation in dilated cardiomyopathy (DCM). We investigated the role of YAP in the pathological process of cardiac matrix remodeling. A classic model of DCM was established in BALB/c mice by immunization with porcine cardiac myosin. Cardiac fibroblasts were isolated from neonatal Sprague-Dawley rats by density gradient centrifugation. The expression levels of α-smooth muscle actin (α-SMA) and collagen volume fraction (CVF) were significantly increased in DCM mice. Angiotensin II (Ang II)-mediated YAP activation promoted the proliferation and transdifferentiation of neonatal rat cardiac fibroblasts, and this effect was significantly suppressed in the shRNA YAP + Ang II group compared with the shRNA Control + Ang II group in vitro (2.98±0.34 ×105 vs 5.52±0.82 ×105, P<0.01). Inhibition of endogenous Ang II-stimulated YAP improved the cardiac function by targeting myofibroblast transdifferentiation to attenuate matrix remodeling in vivo. In the valsartan group, left ventricular ejection fraction and fractional shortening were significantly increased compared with the DCM group (52.72±5.51% vs 44.46±3.01%, P<0.05; 34.84±3.85% vs 26.65±3.12%, P<0.01). Our study demonstrated that YAP was a regulator of cardiac myofibroblast differentiation, and regulation of YAP signaling pathway contributed to improve cardiac function of DCM mice, possibly in part by decreasing myofibroblast transdifferentiation to inhibit matrix remodeling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Angiotensina II/farmacologia , Cardiomiopatia Dilatada/fisiopatologia , Transdiferenciação Celular/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Fosfoproteínas/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Western Blotting , Cardiomiopatia Dilatada/patologia , Proteínas de Ciclo Celular , Diferenciação Celular , Modelos Animais de Doenças , Ecocardiografia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Miofibroblastos/fisiologia , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/fisiologia , Ratos , Ratos Sprague-Dawley , Suínos
17.
Sci Rep ; 8(1): 16735, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30425290

RESUMO

The bioactive plant diterpene oridonin displays important pharmacological activities and is widely used in traditional Chinese medicine; however, its molecular mechanism of action is still incompletely described. In vitro and in vivo data have demonstrated anti-tumor activity of oridonin and its ability to interfere with several cell pathways; however, presently only the molecular chaperone HSP70 has been identified as a direct potential target of this compound. Here, using a combination of different proteomic approaches, innovative Cellular Thermal Shift Assay (CETSA) experiments, and classical biochemical methods, we demonstrate that oridonin interacts with Nucleolin, effectively modulating the activity of this multifunctional protein. The ability of oridonin to target Nucleolin and/or HSP70 could account for the bioactivity profile of this plant diterpene. Recently, Nucleolin has attracted attention as a druggable target, as its diverse functions are implicated in pathological processes such as cancer, inflammation, and viral infection. However, up to now, no small molecule as Nucleolin binders has been reported, thus our finding represents the first evidence of Nucleolin modulation by a small inhibitor.


Assuntos
Antineoplásicos/farmacologia , Diterpenos de Caurano/farmacologia , Fosfoproteínas/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Antineoplásicos/metabolismo , Transporte Biológico , Diterpenos de Caurano/metabolismo , Células HeLa , Humanos , Células Jurkat
18.
mBio ; 9(6)2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30401776

RESUMO

The main obstacle to an HIV cure is the transcriptionally inert proviruses that persist in resting CD4 T cells and other reservoirs. None of the current approaches has significantly reduced the size of the viral reservoir. Hence, alternative approaches, such as permanent blocking of viral transcription, to achieve a sustained remission, need urgent attention. To identify cellular factors that may be important for this approach, we sought for host targets that when altered could block HIV transcription and reactivation. Here, we identified splicing factor 3B subunit 1 (SF3B1) as a critical HIV dependency factor required for viral replication. SF3B1 is a splicing factor involved in directing chromatin and nascent gene transcripts to appropriate splice sites. Inhibitors of SF3B1 are currently in development for cancer and have been found to be nontoxic to normal cells compared to malignant cells. Knockdown of SF3B1 abrogated HIV replication in all cell types tested. SF3B1 interacted with viral protein Tat in vitro and in vivo Genetic or pharmacologic inhibition of SF3B1 prevented Tat-mediated HIV transcription and RNA polymerase II association with the HIV promoter. In addition, an inhibitor of SF3B1 prevented HIV reactivation from latency irrespective of the latency-reversing agent used. The data show that SF3B1 is involved in viral transcription and reactivation from latency and may serve as a therapeutic target in the HIV cure efforts.IMPORTANCE The reason why HIV cannot be cured by current therapy is because of viral persistence in resting T cells. One approach to permanent HIV remission that has received less attention is the so-called "block and lock" approach. The idea behind this approach is that the virus could be permanently disabled in patients if viral genome or surrounding chromatin could be altered to silence the virus, thus enabling patients to stop therapy. In this work, we have identified splicing factor 3B subunit 1 (SF3B1) as a potential target for this approach. SF3B1 interacts with the viral protein Tat, which is critical for viral transcription. Inhibition of SF3B1 prevents HIV transcription and reactivation from latency. Since there are preclinical inhibitors for this protein, our findings could pave the way to silence HIV transcription, potentially leading to prolonged or permanent remission.


Assuntos
HIV-1/fisiologia , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Ativação Viral , Latência Viral , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Cromatina/genética , Cicloexilaminas/farmacologia , Células HEK293 , HIV-1/genética , Humanos , Células Jurkat , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/antagonistas & inibidores , Fatores de Processamento de RNA/metabolismo , RNA Interferente Pequeno , Compostos de Espiro/farmacologia , Células THP-1 , Transcrição Genética/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
19.
Cell Physiol Biochem ; 50(4): 1510-1521, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30359987

RESUMO

BACKGROUND/AIMS: Periodic mechanical stress has been shown to promote extracellular matrix (ECM) synthesis and cell migration of nucleus pulposus (NP) cells, however, the mechanisms need to be fully elucidated. The present study aimed to investigate the signal transduction pathway in the regulation of NP cells under periodic mechanical stress. METHODS: Primary rat NP cells were isolated and seeded on glass slides, and then treated in our self-developed periodic stress field culture system. To further explore the mechanisms, data were analyzed by scratch-healing assay, quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, western blotting, and co-immunoprecipitation assay. RESULTS: Under periodic mechanical stress, the mRNA expression of ECM collagen 2A1 (Col2A1) and aggrecan, and migration of NP cells were significantly increased (P < 0.05 for each), associating with increases in the phosphorylation of Src, GIT1, and ERK1/2 (P < 0.05 for each). Pretreatment with the Src inhibitor PP2 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each), while the phosphorylation of GIT1 and ERK1/2 were inhibited. ECM synthesis, cell migration, and phosphorylation of ERK1/2 were inhibited after pretreatment with the small interfering RNA for GIT1 in NP cells under periodic mechanical stress (P < 0.05 for each), whereas the phosphorylation of Src was not affected. Pretreatment with the ERK1/2 inhibitor PD98059 reduced periodic mechanical stress-induced ECM synthesis and cell migration of NP cells (P < 0.05 for each). Co-immunoprecipitation assay showed that there was a direct interaction between Src and GIT1 and between GIT1 and ERK1/2. CONCLUSION: In conclusion, periodic mechanical stress induced ECM expression and migration of NP cells via Src-GIT1-ERK1/2 signaling pathway, playing an important role in regulation of NP cells.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Estresse Mecânico , Quinases da Família src/metabolismo , Agrecanas/metabolismo , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Flavonoides/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Núcleo Pulposo/citologia , Núcleo Pulposo/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores
20.
Cell Physiol Biochem ; 50(3): 1015-1028, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30355914

RESUMO

BACKGROUND/AIMS: The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively, but the mechanisms whereby chondrocytes sense and respond to mechanical stimuli remain to be determined. We explored the question and verified the key role of G protein coupled receptor kinase interacting protein 1 (GIT1) signaling in periodic mechanical stress-induced chondrocyte proliferation. METHODS: Two steps were undertaken in the experiment. In the first step, the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to GIT1 or Src or control scrambled shRNA, or transfected with GIT1 wild-type or GIT1 mutant Y321F, or focal adhesion kinase (FAK) wild-type or FAK mutants Y397F or Y576F/Y577, respectively. Moreover, the cells were pretreated with blocking antibody against integrin ß1 or PP2. Then the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. RESULTS: Periodic mechanical stress significantly induced sustained phosphorylation of GIT1 at Tyr321. Reduction of GIT1 with shRNA targeted to GIT1 and GIT1 mutant Y321F inhibited periodic mechanical stress-promoted chondrocyte proliferation, accompanied by attenuated extracellular signal-regulated kinase (ERK)1/2 and FAK phosphorylation at Tyr576/577. However, activation of Src and FAK-Tyr397 was not prevented upon GIT1 suppression. Furthermore, pretreatment with blocking antibody against integrin ß1, Src-selective inhibitor, PP2, and shRNA targeted to Src blocked GIT1 activation under periodic mechanical stress. In addition, GIT1 phosphorylation at Tyr321 was not reduced upon pretreatment with FAK mutants Y397F or Y576F/Y577 under conditions of periodic mechanical stress. CONCLUSION: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through at least two separate pathways, integrin ß1-Src-GIT1-FAK(Tyr576/577)-ERK1/2, and the other parallel GIT1-independent integrin ß1-FAK(Tyr397)-ERK1/2.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Estresse Mecânico , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Integrina beta1/imunologia , Integrina beta1/metabolismo , Mutagênese Sítio-Dirigida , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Quinases da Família src/metabolismo
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