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1.
Oncogene ; 38(39): 6615-6629, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31371779

RESUMO

Oncogenic KIT or PDGFRA tyrosine kinase mutations are compelling therapeutic targets in most gastrointestinal stromal tumors (GISTs), and the KIT inhibitor, imatinib, is therefore standard of care for patients with metastatic GIST. However, some GISTs lose expression of KIT oncoproteins, and therefore become KIT-independent and are consequently resistant to KIT-inhibitor drugs. We identified distinctive biologic features in KIT-independent, imatinib-resistant GISTs as a step towards identifying drug targets in these poorly understood tumors. We developed isogenic GIST lines in which the parental forms were KIT oncoprotein-dependent, whereas sublines had loss of KIT oncoprotein expression, accompanied by markedly downregulated expression of the GIST biomarker, protein kinase C-theta (PRKCQ). Biologic mechanisms unique to KIT-independent GISTs were identified by transcriptome sequencing, qRT-PCR, immunoblotting, protein interaction studies, knockdown and expression assays, and dual-luciferase assays. Transcriptome sequencing showed that cyclin D1 expression was extremely low in two of three parental KIT-dependent GIST lines, whereas cyclin D1 expression was high in each of the KIT-independent GIST sublines. Cyclin D1 inhibition in KIT-independent GISTs had anti-proliferative and pro-apoptotic effects, associated with Rb activation and p27 upregulation. PRKCQ, but not KIT, was a negative regulator of cyclin D1 expression, whereas JUN and Hippo pathway effectors YAP and TAZ were positive regulators of cyclin D1 expression. PRKCQ, JUN, and the Hippo pathway coordinately regulate GIST cyclin D1 expression. These findings highlight the roles of PRKCQ, JUN, Hippo, and cyclin D1 as oncogenic mediators in GISTs that have converted, during TKI-therapy, to a KIT-independent state. Inhibitors of these pathways could be effective therapeutically for these now untreatable tumors.


Assuntos
Ciclina D1/fisiologia , Tumores do Estroma Gastrointestinal/genética , Proteínas Proto-Oncogênicas c-kit/genética , Antineoplásicos/uso terapêutico , Proliferação de Células/fisiologia , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/metabolismo , Humanos , Proteína Quinase C-theta/fisiologia , Inibidores de Proteínas Quinases/uso terapêutico
2.
Artif Cells Nanomed Biotechnol ; 47(1): 1984-1994, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31311343

RESUMO

Recently, we showed that parathyroid hormone-like hormone (PTHLH), a cytokine-like polyprotein, is critical for extracellular matrix (ECM) deposition through the activation of hepatic stellate cells (HSCs). Here, we show that N-terminal PTHLH is secreted into the supernatant of injured hepatocytes, its expression is positively correlated with liver fibrosis severity based on mice liver biopsies, and it is primarily expressed in the cytoplasm of hepatocytes along the fibrous septa of fibrotic livers. PTHLH overexpression in mice was achieved through adeno-associated virus-mediated gene delivery (AAV9-PTHLH), and liver fibrosis was induced with carbon tetrachloride (CCl4). We observed that AAV9-PTHLH induced spontaneous development of liver fibrosis and increased sensitivity to CCl4. PTHLH increased Hedgehog (Hh) pathway activation in a PTH1R-dependent manner, and the effect of PTHLH was primarily mediated by protein kinase C (PKC) θ. PTHLH-mediated PTH1R-PKC θ pathway activation is a key event in the profibrotic Hh-dependent activation of HSCs.


Assuntos
Proteínas Hedgehog/metabolismo , Células Estreladas do Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Transdução de Sinais , Animais , Células Estreladas do Fígado/metabolismo , Masculino , Proteína Quinase C-theta/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Regulação para Cima
3.
Inflammation ; 42(6): 1980-1989, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31297750

RESUMO

Acute lung injury (ALI)/acute respiratory distress syndrome is characterized by increased pulmonary inflammation, where T helper 17 (Th17) cells play an important regulatory role. Notch signaling critically regulates Th17 differentiation and is known to be linked with proximal T cell by protein kinase C theta (PKCθ). We hypothesized that PKCθ inhibition could attenuate ALI by suppressing Th17 response via the Notch signaling pathway. Male C57BL/6 mice were treated with phosphate-buffered saline (PBS), lipopolysaccharide (LPS), LPS and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT, a Notch signaling inhibitor), or LPS and PKCθ inhibitor (PI), and the bronchoalveolar lavage fluid (BALF), blood, and lung tissues were harvested at 48 h after the LPS challenge. CD4+ T cells were treated with DAPT or PI and harvested after 72 h. PKCθ inhibition markedly attenuated pathological changes and decreased the wet to dry weight ratio of the mouse lungs. The total cell and neutrophil counts, tumor necrosis factor-α (TNF- α) in BALF, myeloperoxidase activity in lung tissue, and the leukocyte count in whole blood were markedly reduced by PKCθ inhibition. The concentration of interleukin (IL)-17 and IL-22 in BALF, and the percentage of CD4+IL-17A+ T cells in the lungs were significantly downregulated by PKCθ inhibition. A similar trend was observed for the expression of retinoic acid-related orphan receptor gamma t and IL-23 receptor after PKCθ inhibition accompanied with inactivation of the Notch signaling pathway in vivo and in vitro. Collectively, these data demonstrated that PKCθ inhibition protects against LPS-induced ALI by suppressing the differentiation and pathogenicity of Th17, at least partially, through a Notch-dependent mechanism.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Proteína Quinase C-theta/antagonistas & inibidores , Receptores Notch/metabolismo , Transdução de Sinais , Células Th17/efeitos dos fármacos , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Diferenciação Celular , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Th17/imunologia , Células Th17/patologia
4.
Int Immunopharmacol ; 74: 105664, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31233937

RESUMO

Lysophosphatidic acid (LPA) is a multifunctional phospholipid. Osteocytes are the most abundant cells in bone and can orchestrate bone formation and resorption, in part by producing cytokines that regulate osteoblast and osteoclast differentiation and activity. Interleukin (IL)-6 and IL-8 are two important cytokines that have potent effects on bone fracture healing. Previous studies suggest that platelet-derived LPA may influence fracture healing by inducing osteocyte dendrite outgrowth. However, the biological mechanism through which LPA induces cytokine production in osteocytes is poorly understood. In this study, we report that LPA markedly enhanced IL-6 and CXCL15 (mouse homologue of human IL-8) production in MLO-Y4 cells and that this enhancement was suppressed by the LPA1/3-selective antagonist Ki16425, the Gi/o protein inhibitor PTX or the protein kinase C (PKC) inhibitor sotrastaurin. We also observed that of all the PKC isoform targets of sotrastaurin, only PKCθ was activated by LPA in MLO-Y4 cells and that this activation was blocked by sotrastaurin, Ki16425 or PTX. Taken together, the results of the present study demonstrate that LPA may be a potent inducer of IL-6 and CXCL15 production in MLO-Y4 cells and that this induction is associated with the activation of LPA1, Gi/o protein and the PKCθ pathway. These findings may help us better understand the mechanism of fracture healing and contribute to the treatment of bone damage.


Assuntos
Quimiocinas CXC/metabolismo , Interleucina-6/metabolismo , Lisofosfolipídeos/farmacologia , Animais , Linhagem Celular , Quimiocinas CXC/genética , Interleucina-6/genética , Camundongos , Proteína Quinase C-theta/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
Cell Commun Signal ; 17(1): 56, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138259

RESUMO

BACKGROUND: The protein kinase C theta (PKCθ) has an important and non-redundant function downstream of the antigen receptor and co-receptor complex in T lymphocytes. PKCθ is not only essential for activation of NF-κB, AP-1 and NFAT and subsequent interleukin-2 expression, but also critical for positive selection and development of regulatory T lymphocytes in the thymus. Several domains regulate its activity, such as a pseudosubstrate sequence mediating an auto-inhibitory intramolecular interaction, the tandem C1 domains binding diacylglycerol, and phosphorylation at conserved tyrosine, threonine as well as serine residues throughout the whole length of the protein. To address the importance of the variable domain V1 at the very N-terminus, which is encoded by exon 2, a mutated version of PKCθ was analyzed for its ability to stimulate T lymphocyte activation. METHODS: T cell responses were analyzed with promoter luciferase reporter assays in Jurkat T cells transfected with PKCθ expression constructs. A mouse line expressing mutated instead of wild type PKCθ was analyzed in comparison to PKCθ-deficient and wild type mice for thymic development and T cell subsets by flow cytometry and T cell activation by quantitative RT-PCR, luminex analysis and flow cytometry. RESULTS: In cell lines, the exon 2-replacing mutation impaired the transactivation of interleukin-2 expression by constitutively active mutant form of PKCθ. Moreover, analysis of a newly generated exon 2-mutant mouse line (PKCθ-E2mut) revealed that the N-terminal replacement mutation results in an hypomorph mutant of PKCθ combined with reduced PKCθ protein levels in CD4+ T lymphocytes. Thus, PKCθ-dependent functions in T lymphocytes were affected resulting in impaired thymic development of single positive T lymphocytes in vivo. In particular, there was diminished generation of regulatory T lymphocytes. Furthermore, early activation responses such as interleukin-2 expression of CD4+ T lymphocytes were significantly reduced even though cell viability was not affected. Thus, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. CONCLUSION: Taken together, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. Both our in vitro T cell culture experiments and ex vivo analyses of a PKCθ-E2-mutant mouse line independently validate the importance of PKCθ downstream of the antigen-receptor complex for activation of CD4+ T lymphocytes.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Ativação Linfocitária , Mutação , Proteína Quinase C-theta/genética , Animais , Células HEK293 , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Proteína Quinase C-theta/metabolismo
6.
J Dig Dis ; 20(5): 243-247, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30828974

RESUMO

OBJECTIVE: Recent genome-wide association studies have demonstrated that rs2236379 in PRKCQ is a novel significant locus for Crohn's disease (CD). However, the association has not been replicated in any populations. We therefore aimed to investigate the prevalence of the PRKCQ rs2236379 variant in the Chinese Han population and evaluate whether the genetic variant of PRKCQ confers susceptibility to CD and is associated with its clinical characteristics. METHODS: A total of 283 patients with CD and 381 healthy controls were enrolled. Genomic DNA was extracted from their whole blood samples and polymerase chain reaction-restriction fragment length polymorphism was used for genotyping. The association between PRKCQ polymorphisms and susceptibility to CD, and between genotypes and clinical phenotypes was analyzed. RESULTS: A higher frequency of the T allele was discovered in CD patients than in healthy controls (P = 0.027). A significant difference in the distribution of the TT and CT/CC genotypes was observed between CD patients and controls (P = 0.024). The TT genotype showed a significant association with susceptibility to CD (odds ratio 1.647, 95% confidence interval: 1.088-2.574, P = 0.019). Patients with CD with the rs2236379 TT mutant risk genotype were most likely to exhibit perianal disease (P = 0.044). CONCLUSIONS: Our research revealed an association between the PRKCQ rs2236379 (C>T) and CD. The TT homozygous mutation increased the risk of developing CD and may contribute to perianal disease.


Assuntos
Doença de Crohn/genética , Mutação de Sentido Incorreto , Proteína Quinase C-theta/genética , Adulto , Estudos de Casos e Controles , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Fragmento de Restrição , Adulto Jovem
7.
Bioorg Med Chem ; 27(5): 790-799, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30704835

RESUMO

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and has therapeutic potential for T cell-mediated diseases such as transplant rejection and rheumatoid arthritis. PKCθ inhibitors have emerged as effective immunomodulative agents for the prevention of transplant rejection. We previously reported that the 2,4-diamino-5-cyanopyrimidine derivative 2 was a potent PKCθ inhibitor; however, it exhibited CYP3A4 time-dependent inhibition (TDI). Here, we report the structural modification of compound 2 into 34 focusing on mitigating CYP3A4 TDI. Compound 34 exhibited potent in vitro activity with mitigated CYP3A4 TDI and efficacy in vivo transplant model.


Assuntos
Diaminas/farmacologia , Proteína Quinase C-theta/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Animais , Inibidores do Citocromo P-450 CYP3A/síntese química , Inibidores do Citocromo P-450 CYP3A/farmacocinética , Inibidores do Citocromo P-450 CYP3A/farmacologia , Diaminas/síntese química , Diaminas/farmacocinética , Descoberta de Drogas , Interações Medicamentosas , Feminino , Rejeição de Enxerto/prevenção & controle , Haplorrinos , Humanos , Células Jurkat , Microssomos Hepáticos/metabolismo , Midazolam/farmacologia , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Ratos Endogâmicos ACI , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Relação Estrutura-Atividade
8.
Fish Shellfish Immunol ; 87: 788-795, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30716520

RESUMO

Haemorrhagic disease caused by grass carp reovirus (GCRV) can result in large-scale death of young grass carp, leading to irreparable economic losses that seriously affect large-scale breeding. Protein kinase C (PKC, also known as PRKC) represents a family of serine/threonine protein kinases that includes multiple isozymes in many species. Among these, PKC-θ (PKC theta, also written as PRKCQ) is a novel isoform, mainly expressed in T cells, that is known to be involved in immune system function in mammals. To date, no research on immunological functions of fish Pkc-θ has been reported. To address this issue, we cloned the grass carp pkc-θ gene. Phylogenetic and syntenic analysis showed that this gene is the most evolutionarily conserved relative to zebrafish. Real-time quantitative PCR (RT-qPCR) indicated that pkc-θ was expressed at high levels in the gills and spleen of healthy grass carp. Infection with GCRV down regulated pkc-θ expression in the gills and spleen. Gene products that function upstream and downstream of pkc-θ were up regulated in the gill, but were down-regulated in the spleen. These results suggest that direct or indirect targeting of pkc-θ by GCRV may help the virus evade host immune defences in the spleen. Phorbol ester (PMA) treatment of Jurkat T cells induced translocation of grass carp Pkc-θ from the cytoplasm to the plasma membrane. This response to PMA suggests evolutionary conservation of an immune response function in fish Pkc-θ, as well as conservation of its sequence and structural domains. This study expanded our knowledge of the fish PKC gene family, and explored the role of pkc-θ in function of the grass carp immune system, providing new insights which may facilitate further studies of its biological functions.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteína Quinase C-theta/genética , Proteína Quinase C-theta/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Proteína Quinase C-theta/química , Distribuição Aleatória , Reoviridae/fisiologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/veterinária , Alinhamento de Sequência/veterinária
9.
Bioorg Chem ; 82: 33-40, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30268972

RESUMO

Six new (1-6) and nine known (7-15) staurosporine derivatives were isolated from the rice solid fermentation of the marine-derived Streptomyces sp. NB-A13. The structures of the new staurosporine derivatives were established by extensive spectroscopic data interpretation. The absolute configurations of 1 and 2 were assigned by quantum chemical calculations of the electronic circular dichroism (ECD) spectra. All of these compounds were screened for their cytotoxic activities against PC-3 and SW-620 cell lines. Compound 7 exhibited stronger inhibitory activity against SW-620 cell lines than the positive control staurosporine (25.10 nM), with IC50 values of 9.99 nM. Moreover, compounds 1-5, 8-13 and 15 also showed significant cytotoxicities with IC50 values ranging from 0.02 to 16.60 µM, while 6 exhibited no cytotoxic potency. Additionally, compounds 1-7 were also tested for enzyme inhibition activities of Protein kinase C theta (PKC-θ), and showed activity with IC50 values ranging from 0.06 to 9.43 µM except for compound 6, which has no inhibition activity.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Streptomyces/química , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Proteína Quinase C-theta/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/isolamento & purificação , Estaurosporina/isolamento & purificação , Estereoisomerismo , Relação Estrutura-Atividade
10.
Fish Shellfish Immunol ; 84: 1100-1107, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30408601

RESUMO

A primitive adaptive immune system has recently been suggested to be present in a basal chordate amphioxus (Branchiostoma belcheri, Bb), making it an ideal model for studying the origin of adaptive immune. The novel protein kinase C isoform PKC-θ, but not its closest isoform PKC-δ, plays a critical role for mammalian T-cell activation via translocation to immunological synapse (IS) mediated by a unique PKC-θ V3 domain containing one PxxP motif. To understand the evolution of this unique PKC-θ V3 domain and the primitive adaptive immune system in amphioxus, we comparatively studied the orthologs of PKC-δ and -θ from amphioxus and other species. Phylogenetic analysis showed BbPKC-δ/θ to be the common ancestor of vertebrate PKC-δ and PKC-θ, with a V3 domain containing two PxxP motifs. One motif is conserved in both zebrafish and mammalian PKC-θ but is absent in PKC-δ V3 domain of these species, and has already emerged in drosophila PKC-δ. The other non-conserved motif emerged in BbPKC-δ/θ, and only retained in Danio rerio PKC-δ (DrPKC-δ) but lost in mammalian PKC-δ and -θ. Comparative analyses of the sequence and function of BbPKC-δ/θ, DrPKC-δ, DrPKC-θ and Homo sapiens PKC-θ (HsPKC-θ) in IS translocation and T-cell receptor (TCR)-induced NF-κB activation revealed that retention of the conserved PxxP motif and loss of the non-conserved PxxP motif in mammalian PKC-θ and loss of both PxxP motifs in mammalian PKC-δ accomplish the unique function of PKC-θ in T cells. Together, this study suggests an evolutionary mechanism for PKC-θ unique V3 and reveals BbPKC-δ/θ is the common ancestor of PKC-δ and -θ with a functional proto-V3 domain, supplying new evidence for the existence of primitive adaptive immune system in amphioxus.


Assuntos
Imunidade Adaptativa/genética , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Anfioxos/genética , Anfioxos/imunologia , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/imunologia , Proteína Quinase C-theta/genética , Proteína Quinase C-theta/imunologia , Sequência de Aminoácidos , Animais , Perfilação da Expressão Gênica/veterinária , Anfioxos/enzimologia , Filogenia , Proteína Quinase C-delta/química , Proteína Quinase C-theta/química , Alinhamento de Sequência/veterinária
11.
Mol Vis ; 25: 834-842, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31908401

RESUMO

Purpose: The PRKCQ and REL genes are said to be associated with multiple autoimmune diseases. This study investigated the association between these genes and Vogt-Koyanagi-Harada (VKH) syndrome in Han Chinese. Methods: A two-stage case-control study was performed on three single nucleotide polymorphisms ([SNPs] rs4750316, rs11258747, and rs947474) of the PRKCQ gene and three SNPs (rs842647, rs702873, and rs13031237) of the REL gene using PCR-restriction fragment length polymorphism (PCR-RFLPs) in a total of 859 patients with VKH syndrome and 1,542 healthy controls. Variables such as extraocular presentations were assessed. The data were analyzed using chi-square analysis, and corrected for multiple comparisons with the Bonferroni method. Results: We found a decreased frequency of the GC genotype and the C allele of rs4750316 in patients with VKH syndrome when the GG genotype or G allele was used as a reference, respectively (GC genotype: P =2.45e-10, odds ratio [OR]=0.37, 95% confidence interval [CI]=0.28-0.51; C allele: P=8.79e-10, OR=0.41, 95% CI=0.31-0.55). The genotypic and allelic frequencies of rs11258747, rs947474, rs842647, rs702873, and rs13031237 were not statistically significantly different between patients with VKH syndrome and controls. Stratification analysis indicated that the PRKCQ rs4750316 polymorphism was associated with patients with VKH syndrome experiencing headache, alopecia, poliosis, tinnitus, and dysacusia, but no statistically significant association of the other five SNPs was found. Conclusions: The PRKCQ rs4750316 polymorphism may be a susceptibility factor for VKH syndrome pathogenesis and extraocular presentations, indicating that PRKCQ may be involved in the pathogenesis and extraocular presentations of VKH syndrome through the T-cell receptor (TCR) signaling pathway.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Grupos Étnicos/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genética , Proteína Quinase C-theta/genética , Síndrome Uveomeningoencefálica/genética , Adulto , Estudos de Casos e Controles , Feminino , Frequência do Gene/genética , Humanos , Masculino
12.
Bioorg Med Chem ; 26(20): 5499-5509, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30274941

RESUMO

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and is an attractive target for the treatment of T cell-mediated diseases such as transplant rejection and autoimmune disease. To identify PKCθ inhibitors, we focused on the 2,6-diamino-3-carbamoyl-5-cyanopyrazine derivative 2, which exhibited moderate PKCθ inhibitory activity. Optimization of 2 identified the 2,4-diamino-5-cyanopyrimidine derivative 16c, which exhibited potent PKCθ inhibitory activity and showed good selectivity against other PKC isozymes. Compound 16c prolonged graft survival in an in vivo rat heterotopic cardiac transplant model.


Assuntos
Rejeição de Enxerto/prevenção & controle , Transplante de Coração/métodos , Proteína Quinase C-theta/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/química , Pirimidinas/uso terapêutico , Animais , Feminino , Rejeição de Enxerto/metabolismo , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante de Coração/efeitos adversos , Humanos , Simulação de Acoplamento Molecular , Proteína Quinase C-theta/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Ratos Sprague-Dawley , Transplante Homólogo/efeitos adversos , Transplante Homólogo/métodos
13.
J Biol Chem ; 293(51): 19771-19784, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30352872

RESUMO

SEC2, a major histocompatibility complex class II (MHC II)-dependent T-cell mitogen, binds MHC II and T-cell receptor (TCR) Vßs to induce effective co-stimulating signals for clonal T-cell expansion. We previously characterized a SEC2 mutant with increased recognition of TCR Vßs, ST-4, which could intensify NF-κB signaling transduction, leading to IL-2 production and T-cell activation. In this study, we found that in contrast to SEC2, ST-4 could induce murine CD4+ T-cell proliferation in a Vß8.2- and Vß8.3-specific manner in the absence of MHC II+ antigen-presenting cells (APCs). Furthermore, although IL-2 secretion in response to either SEC2 or ST-4 stimulation was accompanied by up-regulation of protein kinase Cθ (PKCθ), inhibitor of κB (IκB), α and ß IκB kinase (IKKα/ß), IκBα, and NF-κB in mouse splenocytes, only ST-4 could activate CD4+ T cells in the absence of MHC II+ APCs through the PKCθ/NF-κB signaling pathway. The PKCθ inhibitor AEB071 significantly suppressed SEC2/ST-4-induced T-cell proliferation, CD69 and CD25 expression, and IL-2 secretion with or without MHC II+ APCs. Further, SEC2/ST-4-induced changes in PKCθ/NF-κB signaling were significantly relieved by AEB071 in a dose-dependent manner. Using Lck siRNA, we found that Lck controlled SEC2/ST-4-induced phosphorylation of PKCθ. We also demonstrated that the IL-2R/STAT5 pathway is essential for SEC2/ST-4-induced T-cell activation. Collectively, our data demonstrate that an enhanced ST-4-TCR interaction can compensate for lack of MHC II and stimulate MHC II-free CD4+ T-cell proliferation via PKCθ/NF-κB and IL-2R/STAT5 signaling pathways. Compared with SEC2, intensified PKCθ/NF-κB and IL-2R/STAT5 signals induced by ST-4 lead to enhanced T-cell activation. The results of this study will facilitate better understanding of TCR-based immunotherapies for cancer.


Assuntos
Enterotoxinas/genética , Ativação Linfocitária , Mutação , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Animais , Proliferação de Células , Enterotoxinas/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Fosforilação , Proteína Quinase C-theta/metabolismo , Receptores de Interleucina-2/metabolismo , Fator de Transcrição STAT5/metabolismo , Baço/imunologia , Linfócitos T/citologia
14.
Sci Adv ; 4(9): eaat5401, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30214937

RESUMO

Retinoic-acid-receptor-related orphan nuclear receptor γt (RORγt) controls the transcription of interleukin-17A (IL-17A), which plays critical roles in the pathogenesis of autoimmune diseases. Severity of several human autoimmune diseases is correlated with frequencies of germinal center kinase-like kinase (GLK) (also known as MAP4K3)-overexpressing T cells; however, the mechanism of GLK overexpression-induced autoimmunity remains unclear. We report the signal transduction converging on aryl hydrocarbon receptor (AhR)-RORγt interaction to activate transcription of the IL-17A gene in T cells. T cell-specific GLK transgenic mice spontaneously developed autoimmune diseases with selective induction of IL-17A in T cells. In GLK transgenic T cells, protein kinase Cθ (PKCθ) phosphorylated AhR at Ser36 and induced AhR nuclear translocation. AhR also interacted with RORγt and transported RORγt into the nucleus. IKKß (inhibitor of nuclear factor κB kinase ß)-mediated RORγt Ser489 phosphorylation induced the AhR-RORγt interaction. T cell receptor (TCR) signaling also induced the novel RORγt phosphorylation and subsequent AhR-RORγt interaction. Collectively, TCR signaling or GLK overexpression induces IL-17A transcription through the IKKß-mediated RORγt phosphorylation and the AhR-RORγt interaction in T cells. Our findings suggest that inhibitors of GLK or the AhR-RORγt complex could be used as IL-17A-blocking agents for IL-17A-mediated autoimmune diseases.


Assuntos
Doenças Autoimunes/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Quinase I-kappa B/metabolismo , Interleucina-17/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Hidrocarboneto Arílico/imunologia , Animais , Doenças Autoimunes/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Feminino , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Células Jurkat , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Proteína Quinase C-theta/genética , Proteína Quinase C-theta/metabolismo , Multimerização Proteica , Transporte Proteico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo
15.
J Clin Invest ; 128(10): 4604-4621, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30106752

RESUMO

Regulatory T cells (Tregs) are critical for maintaining immune homeostasis. However, current Treg immunotherapies do not optimally treat inflammatory diseases in patients. Understanding the cellular processes that control Treg function may allow for the augmentation of therapeutic efficacy. In contrast to activated conventional T cells, in which protein kinase C-θ (PKC-θ) localizes to the contact point between T cells and antigen-presenting cells, in human and mouse Tregs, PKC-θ localizes to the opposite end of the cell in the distal pole complex (DPC). Here, using a phosphoproteomic screen, we identified the intermediate filament vimentin as a PKC-θ phospho target and show that vimentin forms a DPC superstructure on which PKC-θ accumulates. Treatment of mouse Tregs with either a clinically relevant PKC-θ inhibitor or vimentin siRNA disrupted vimentin and enhanced Treg metabolic and suppressive activity. Moreover, vimentin-disrupted mouse Tregs were significantly better than controls at suppressing alloreactive T cell priming in graft-versus-host disease (GVHD) and GVHD lethality, using a complete MHC-mismatch mouse model of acute GVHD (C57BL/6 donor into BALB/c host). Interestingly, vimentin disruption augmented the suppressor function of PKC-θ-deficient mouse Tregs. This suggests that enhanced Treg activity after PKC-θ inhibition is secondary to effects on vimentin, not just PKC-θ kinase activity inhibition. Our data demonstrate that vimentin is a key metabolic and functional controller of Treg activity and provide proof of principle that disruption of vimentin is a feasible, translationally relevant method to enhance Treg potency.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Filamentos Intermediários/imunologia , Ativação Linfocitária , Linfócitos T Reguladores/imunologia , Vimentina/imunologia , Animais , Células Apresentadoras de Antígenos/patologia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Humanos , Filamentos Intermediários/genética , Filamentos Intermediários/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteína Quinase C-theta/genética , Proteína Quinase C-theta/imunologia , Linfócitos T Reguladores/patologia , Vimentina/genética
16.
BMC Anesthesiol ; 18(1): 87, 2018 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-30021515

RESUMO

BACKGROUND: Interleukin 2 (IL-2) influences the development and severity of pain due to its antinociceptive and immunomodulatory effects. Its production is influenced by the increased expression of c-Cbl (Casitas B-lineage lymphoma proto-oncogene) and Cbl-b E3 ubiquitin ligases. We evaluated the effects on IL-2-mediated changes in c-Cbl and Cbl-b expression in a rat model of chronic neuropathic pain. METHODS: Peripheral neuropathy was induced in adult male Sprague-Dawley rats weighing 250-300 g by chronic spinal nerve ligation. Half of the spinal cord ipsilateral to the nerve injury was harvested at 1, 3, and 6 weeks, and the expression levels of IL-2, c-Cbl, Cbl-b, phospholipase C-γ1 (PLC-γ1), ZAP70, and protein kinase Cθ (PKCθ), as well as ubiquitin conjugation, were evaluated. RESULTS: Total IL-2 mRNA levels were significantly decreased at 3 and 6 weeks after nerve injury compared to those in sham-operated rats. The mRNA levels of c-Cbl and Cbl-b, as well as the level of ubiquitin conjugation, were significantly increased at 3 and 6 weeks. In contrast, the levels of phosphorylated ZAP70 and PLC-γ1 were decreased at 3 and 6 weeks after spinal nerve ligation. Ubiquitination of PLC-γ1 and PKCθ was increased at 3 and 6 weeks. CONCLUSIONS: Our results suggest that ubiquitin and the E3 ubiquitin ligases c-Cbl and Cbl-b function as neuroimmune modulators in the subacute phase of neuropathic pain after nerve injury.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Interleucina-2/biossíntese , Doenças do Sistema Nervoso Periférico/metabolismo , Proteínas Proto-Oncogênicas c-cbl/biossíntese , Ubiquitina-Proteína Ligases/biossíntese , Animais , Ligadura , Masculino , Fosfolipase C gama/biossíntese , Proteína Quinase C-theta/biossíntese , Ratos , Medula Espinal/metabolismo , Nervos Espinhais/lesões , Ubiquitina/metabolismo , Ubiquitinação , Proteína-Tirosina Quinase ZAP-70/biossíntese
17.
Circulation ; 138(21): 2395-2412, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-29991487

RESUMO

BACKGROUND: Although the role of thrombin in atherothrombosis is well studied, its role in the pathogenesis of diet-induced atherosclerosis is not known. METHODS: Using a mouse model of diet-induced atherosclerosis and molecular biological approaches, here we have explored the role of thrombin and its G protein-coupled receptor signaling in diet-induced atherosclerosis. RESULTS: In exploring the role of G protein-coupled receptor signaling in atherogenesis, we found that thrombin triggers foam cell formation via inducing CD36 expression, and these events require Par1-mediated Gα12-Pyk2-Gab1-protein kinase C (PKC)θ-dependent ATF2 activation. Genetic deletion of PKCθ in apolipoprotein E (ApoE)-/- mice reduced Western diet-induced plaque formation. Furthermore, thrombin induced Pyk2, Gab1, PKCθ, and ATF2 phosphorylation, CD36 expression, and foam cell formation in peritoneal macrophages of ApoE-/- mice. In contrast, thrombin only stimulated Pyk2 and Gab1 but not ATF2 phosphorylation or its target gene CD36 expression in the peritoneal macrophages of ApoE-/-:PKCθ-/- mice, and it had no effect on foam cell formation. In addition, the aortic root cross-sections of Western diet-fed ApoE-/- mice showed increased Pyk2, Gab1, PKCθ, and ATF2 phosphorylation and CD36 expression as compared with ApoE-/-:PKCθ-/- mice. Furthermore, although the monocytes from peripheral blood and the aorta of Western diet-fed ApoE-/- mice were found to contain more of Ly6Chi cells than Ly6Clo cells, the monocytes from Western diet-fed ApoE-/-:PKCθ-/- mice were found to contain more Ly6Clo cells than Ly6Chi cells. It is interesting to note that the Ly6Chi cells showed higher CD36 expression with enhanced capacity to form foam cells as compared with Ly6Clo cells. CONCLUSIONS: These findings reveal for the first time that thrombin-mediated Par1-Gα12 signaling via targeting Pyk2-Gab1-PKCθ-ATF2-dependent CD36 expression might be playing a crucial role in diet-induced atherogenesis.


Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Aterosclerose/patologia , Antígenos CD36/metabolismo , Proteína Quinase C-theta/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Aterosclerose/veterinária , Antígenos CD36/antagonistas & inibidores , Antígenos CD36/genética , Diferenciação Celular/efeitos dos fármacos , Células Espumosas/citologia , Células Espumosas/metabolismo , Quinase 2 de Adesão Focal/antagonistas & inibidores , Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Proteína Quinase C-theta/deficiência , Proteína Quinase C-theta/genética , Células RAW 264.7 , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Trombina/farmacologia , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
18.
Cell Immunol ; 331: 137-145, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29954581

RESUMO

Platelets modulate the immune system following injury by interacting with CD4+ T regulatory cells (CD4+ Tregs). The underlying mechanisms remain unsolved. We hypothesize paracrine interactions via Tumor necrosis factor-alpha (TNFα)-, Toll like receptor-4 (TLR4)-, and Interleukin-10 (IL-10). In the murine burn injury model, CD4+ Treg activation pathways were selectively addressed using TNFR2-, TLR4- and IL-10-deficient mice. The CD4+ Treg signalling molecule PKC-θ was analyzed using phospho-flow cytometry to detect rapid cell activation. Thromboelastometry (ROTEM®) was used to assess platelet activation. Injury induced significant early activation of CD4+ Tregs, disruption of TNFR2 and TLR4 activation pathways resulted in lower activity. The disruption of IL-10 crosstalk had no significant impact. Selective disruption of paracrine interactions is associated with changes in posttraumatic hemostasis parameters. TNFR2- and TLR4-dependent pathways modulate the activation of CD4+ Tregs following trauma. In contrast, we did not observe a role of IL-10 in the posttraumatic activation of CD4+ Tregs. ONE SENTENCE SUMMARY: TLR4- and TNFR2-dependent mechanisms, but not IL-10-dependent pathways, modulate the anti-inflammatory response of CD4+ Tregs following trauma.


Assuntos
Queimaduras/imunologia , Interleucina-10/imunologia , Ativação Linfocitária/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Linfócitos T Reguladores/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Queimaduras/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação Plaquetária/imunologia , Proteína Quinase C-theta/imunologia , Proteína Quinase C-theta/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Reguladores/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
19.
Bioorg Med Chem Lett ; 28(10): 1964-1971, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29636220

RESUMO

Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines.


Assuntos
Proteína Quinase C-theta/metabolismo , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Concentração Inibidora 50 , Interleucina-2/metabolismo , Camundongos , Camundongos Knockout , Simulação de Acoplamento Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologia , Relação Estrutura-Atividade , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
20.
Bioorg Chem ; 78: 46-57, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29533214

RESUMO

Recent developments in the literature have demonstrated that curcumin exhibit antioxidant properties supporting its anti-inflammatory, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Despite the valuable findings of curcumin against different cancer cells, the clinical use of curcumin in cancer treatment is limited due to its extremely low aqueous solubility and instability, which lead to poor in vivo bioavailability and limited therapeutic effects. We therefore focused in the present study to evaluate the anti-tumor potential of curcumin analogues on the human breast carcinoma cell lines MDA-MB-231 and MCF-7, as well as their effects on non-tumorigenic normal breast epithelial cells (MCF-10). The IC50 values of curcumin analogue J1 in these cancer cell lines were determined to be 5 ng/ml and 10 ng/ml, in MDA-MB-231 and MCF-7 cells respectively. Interestingly, at these concentrations, the J1 did not affect the viability of non-tumorigenic normal breast epithelial cells MCF-10. Furthermore, we found that J1 strongly induced growth arrest of these cancer cells by modulating the mitochondrial membrane potentials without significant effect on normal MCF-10 cells using JC-1 staining and flow cytometry analysis. Using annexin-V/PI double staining assay followed by flow cytometry analysis, we found that J1 robustly enhanced the induction of apoptosis by increasing the activity of caspases in MDA-MB-231 and MCF-7 cancer cells. In addition, treatment of breast cancer cells with J1 revealed that, in contrast to the expression of cyclin B1, this curcumin analogue vigorously decreased the expression of cyclin A, CDK2 and cyclin E and subsequently sensitized tumor cells to cell cycle arrest. Most importantly, the phosphorylation of AKT, mTOR and PKC-theta in J1-treated cancer cells was markedly decreased and hence affecting the survival of these cancer cells. Most interestingly, J1-treated cancer cells exhibited a significant inhibition in the activation of RhoA followed by reduction in actin polymerization and cytoskeletal rearrangement in response to CXCL12. Our data reveal the therapeutic potential of the curcumin analogue J1 and the underlying mechanisms to fight breast cancer cells.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Curcumina/análogos & derivados , Curcumina/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Quinase C-theta/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Estrutura Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C-theta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas
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