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1.
Biosci Rep ; 34(4)2014 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-24993645

RESUMO

NO production catalysed by eNOS (endothelial nitric-oxide synthase) plays an important role in the cardiovascular system. A variety of agonists activate eNOS through the Ser1177 phosphorylation concomitant with Thr495 dephosphorylation, resulting in increased ·NO production with a basal level of calcium. To date, the underlying mechanism remains unclear. We have previously demonstrated that perturbation of the AIE (autoinhibitory element) in the FMN-binding subdomain can also lead to eNOS activation with a basal level of calcium, implying that the AIE might regulate eNOS activation through modulating phosphorylation at Thr495 and Ser1177. Here we generated stable clones in HEK-293 (human embryonic kidney 293) cells with a series of deletion mutants in both the AIE (Δ594-604, Δ605-612 and Δ626-634) and the C-terminal tail (Δ14; deletion of 1164-1177). The expression of Δ594-604 and Δ605-612 mutants in non-stimulated HEK-293 cells substantially increased nitrate/nitrite release into the culture medium; the other two mutants, Δ626-634 and Δ1164-1177, displayed no significant difference when compared with WTeNOS (wild-type eNOS). Intriguingly, mutant Δ594-604 showed close correlation between Ser1177 phosphorylation and Thr495 dephosphorylation, and NO production. Our results have indicated that N-terminal portion of AIE (residues 594-604) regulates eNOS activity through coordinated phosphorylation on Ser1177 and Thr495.


Assuntos
Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação/genética , Cálcio/metabolismo , Células Cultivadas , Meios de Cultura/metabolismo , Células HEK293 , Humanos , Mutação/genética , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo
2.
PLoS One ; 7(11): e49232, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23155470

RESUMO

Quiescent cells are considered to be dormant. However, recent studies suggest that quiescent fibroblasts possess active metabolic profile and certain functional characteristics. We previously observed that serum-starved quiescent fibroblasts respond to proinflammatory stimuli by robust expression of cyclooxygenase-2 (COX-2), which declines after the quiescent fibroblasts are driven to proliferation. In this study, we elucidated the underlying signaling and transcriptional mechanism and identified by microarray genes with similar differential expression. By using pharmacological inhibitors coupled with gene silencing, we uncovered the key role of protein kinase C δ (PKCδ) and extracellular signal regulated protein kinase 1/2 (ERK1/2) signaling in mediating COX-2 expression in quiescent cells. Surprisingly, COX-2 expression in proliferative cells was not blocked by PKCδ or ERK1/2 inhibitors due to intrinsic inhibition of PKCδ and ERK1/2 in proliferative cells. Restrained COX-2 transcription in proliferative cells was attributable to reduced NF-κB binding. Microarray analysis identified 35 genes whose expressions were more robust in quiescent than in proliferative cells. A majority of those genes belong to proinflammatory cytokines, chemokines, adhesive molecules and metalloproteinases, which require NF-κB for transcription. Quiescent fibroblasts had a higher migratory activity than proliferative fibroblasts as determined by the transwell assay. Selective COX-2 inhibition reduced migration which was restored by prostaglandin E(2). As COX-2 and inflammatory mediators induce DNA oxidation, we measured 8-hydroxydeoxyguanosine (8-OHdG) in quiescent vs. proliferative fibroblasts. PMA-induced 8-OHdG accumulation was significantly higher in quiescent than in proliferative fibroblasts. These findings indicate that quiescent fibroblasts (and probably other quiescent cells) are at the forefront in mounting inflammatory responses through expression of an array of proinflammatory genes via the PKCδ/ERK1/2 signaling pathway.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fibroblastos/metabolismo , Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Acetofenonas/farmacologia , Benzopiranos/farmacologia , Movimento Celular/efeitos dos fármacos , Cromonas/farmacologia , Ciclo-Oxigenase 2/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Flavonoides/farmacologia , Humanos , Inflamação/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Morfolinas/farmacologia , NF-kappa B/metabolismo , Proteína Quinase C-delta/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia
3.
Proc Natl Acad Sci U S A ; 109(33): 13231-6, 2012 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-22851770

RESUMO

Cyclooxygenase-2 (COX-2) expression is induced by mitogenic and proinflammatory factors. Its overexpression plays a causal role in inflammation and tumorigenesis. COX-2 expression is tightly regulated, but the mechanisms are largely unclear. Here we show the control of COX-2 expression by an endogenous tryptophan metabolite, 5-methoxytryptophan (5-MTP). By using comparative metabolomic analysis and enzyme-immunoassay, our results reveal that normal fibroblasts produce and release 5-MTP into the extracellular milieu whereas A549 and other cancer cells were defective in 5-MTP production. 5-MTP was synthesized from L-tryptophan via tryptophan hydroxylase-1 and hydroxyindole O-methyltransferase. 5-MTP blocked cancer cell COX-2 overexpression and suppressed A549 migration and invasion. Furthermore, i.p. infusion of 5-MTP reduced tumor growth and cancer metastasis in a murine xenograft tumor model. We conclude that 5-MTP synthesis represents a mechanism for endogenous control of COX-2 overexpression and is a valuable lead for new anti-cancer and anti-inflammatory drug development.


Assuntos
Transformação Celular Neoplásica/patologia , Ciclo-Oxigenase 2/metabolismo , Triptofano/análogos & derivados , Acetilserotonina O-Metiltransferasa/metabolismo , Animais , Biocatálise/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Metabolômica , Camundongos , Metástase Neoplásica , Solubilidade/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Triptofano/biossíntese , Triptofano/metabolismo , Triptofano/farmacologia , Triptofano Hidroxilase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Biorheology ; 49(1): 15-25, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22513865

RESUMO

OBJECTIVE: We aimed to elucidate the frequency-specific microcirculatory blood-flow (MBF) effect induced by weak vibration stimulation (VS) in healthy human subjects. METHODS: VS was implemented by a rod (connected to a DC motor) hitting a water-filled bag. VS was applied to the left palm at frequencies near to the heart rate (HR) (n=35; Group A) and 50% higher than the HR (n=20; Group B), and laser-Doppler-flowmetry measurements were made on the back of the left hand (Site 1). A control group without applying VS was also used (n=21). RESULTS: The mean MBF (MMBF), pulsatile MBF, and pulse width at Site 1 only increased significantly in Group A, and the coefficient of variance of the MMBF sequence increased significantly at Site 1 in Groups A and B. CONCLUSION: These results illustrate the VS-induced changes in the regulatory activities of arteriolar openings, and improvement of the MBF near the VS application site in Group A. The improvement in MBF depended on whether the VS frequency was near to, or higher than the HR. The present findings may be pertinent to amelioration of disease induced by an abnormal MBF.


Assuntos
Frequência Cardíaca , Microcirculação , Pele/irrigação sanguínea , Pele/química , Adulto , Humanos , Fluxometria por Laser-Doppler , Masculino , Fenômenos Fisiológicos da Pele , Vibração , Adulto Jovem
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