RESUMO
PIM (proviral integration site) kinases are a distinct class of serine/threonine-specific kinases consisting of PIM1, PIM2 and PIM3. PIM2 is known to function in apoptosis pathways. Expression of PIM2 is highly induced by pro-inflammatory stimuli but the role of PIM2 in the expression of pro-inflammatory cytokines is unclear. In this study, we showed that over-expression of PIM2 in HeLa cells as well as in human umbilical vein endothelial cells enhanced interleukin-1ß (IL-1ß) -induced and tumour necrosis factor-α-induced IL-6 expression, whereas over-expression of a kinase-dead PIM2 mutant had the opposite effect. Studies with small interfering RNA specific to PIM2 further confirmed that IL-6 expression in HeLa cells requires PIM2. To investigate the function of PIM2 further, we generated PIM2-deficient mice. It was found that IL-6 production was significantly decreased from PIM2-deficient spleen cells after stimulation with lipopolysaccharide. Taken together, we demonstrated an important function of PIM2 in controlling the expression of the pro-inflammatory cytokine IL-6. PIM2 inhibitors may be beneficial for IL-6-mediated diseases such as rheumatoid arthritis.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/farmacologia , Interleucina-6/biossíntese , Lipopolissacarídeos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células HeLa , Humanos , Interleucina-10/metabolismo , Interleucina-6/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Baço/citologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/genéticaRESUMO
Epstein-Barr virus-induced gene 3 (EBI3) associates with p28 to form IL-27 and with IL-12p35 to form IL-35. IL-27Ralpha(-/-) mice studies indicate that IL-27 negatively regulates Th17 cell differentiation. However, no EBI3, p28 or p35-deficiency studies that directly address the role of EBI3, p28 or p35 on Th17 cells have been done. Here, we demonstrate that spleen cells derived from EBI3(-/-) mice produce significantly higher levels of IL-17 as well as IL-22 upon stimulation with OVA. In vitro derived EBI3(-/-) Th17 cells also produced significantly higher levels of IL-17 and IL-22 than WT cells. The frequency of IL-17-producing cells was also elevated when EBI3(-/-) cells were cultured under Th17 conditions. In addition, spleen cells from EBI3(-/-) mice immunized with Listeria monocytogenes produced significantly elevated levels of IL-17 and IL-22. Furthermore, the Th17 transcription factor RORgamma t was significantly enhanced in EBI3(-/-) cells. Finally, EBI3(-/-) mice exhibited a reduced bacterial load following an acute challenge with L. monocytogenes or a re-challenge of previously immunized mice, suggesting that EBI3 negatively regulates both innate and adaptive immunity. Taken together, these data provide direct evidence that EBI3 negatively regulates the expression of IL-17, IL-22 and RORgamma t as well as protective immunity against L. monocytogenes.
Assuntos
Regulação da Expressão Gênica , Interleucina-17/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Linfócitos T/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/imunologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica/genética , Interferon gama/sangue , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-17/sangue , Interleucina-17/genética , Listeria monocytogenes/imunologia , Listeriose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Ovalbumina/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Baço/citologia , Baço/microbiologia , Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Interleucina 22RESUMO
The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did not work in vivo upon oral administration. Our current work highlights the further use of rational drug design and molecular modeling to produce a series of lck inhibitors that demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in vivo mouse model of anti-CD3-induced IL-2 production.
Assuntos
Benzimidazóis/síntese química , Inibidores Enzimáticos/síntese química , Imunossupressores/síntese química , Isoquinolinas/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Administração Oral , Animais , Anticorpos Monoclonais/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Complexo CD3/imunologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imunossupressores/química , Imunossupressores/farmacologia , Interleucina-2/antagonistas & inibidores , Interleucina-2/biossíntese , Interleucina-2/sangue , Isoquinolinas/química , Isoquinolinas/farmacologia , Células Jurkat , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
An imidazo[4,5-h]isoquinolin-7,9-dione (1) was identified as an adenosine 5'-triphosphate competitive inhibitor of lck by high throughput screening. Initial structure-activity relationship studies identified the dichlorophenyl ring and the imide NH as important pharmacophores. A binding model was constructed to understand how 1 binds to a related kinase, hck. These results suggested that removing the gem-dimethyl group and flattening the ring would enhance activity. This was realized by converting 1 to the imidazo[4,5-h]isoquinolin-9-one (20), resulting in an 18-fold improvement in potency against lck and a 50-fold increase in potency in a cellular assay.