Acta Pharmacol Sin. 2003 Jul;24(7):637-40. Construction of a recombinant vector based on AAV carrying human endothelial nitric-oxide synthase gene.
He CX, Miao CY, Yao JH, Chen HM, Lu DR, Su DF, Xue JL.
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China.
AIM: To construct an AAV based vector carrying human endothelial nitric-oxide synthase (eNOS) cDNA and study its expression in vitro for future gene therapy. METHODS: eNOS cDNA was inserted into the EcoR I site of pSNAV-1 containing the cytomegalovirus (CMV) promoter and inverted terminal repeat sequences of adeno-associated virus. The constructed vector was transfected into BHK and C2C12 cells. eNOS cDNA and mRNA were detected by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), respectively. RESULTS: By restriction enzyme digestion analysis, it was proved that eNOS cDNA was inserted into pSNAV-1 in a proper direction. PCR detection demonstrated that pSNAV-eNOS was transferred into both BHK and C2C12 cells. RT-PCR analysis showed that these pSNAV-eNOS transfected cells could express eNOS mRNA. CONCLUSION: pSNAV-eNOS was successfully constructed with the ability to express human eNOS mRNA in cultured mammalian cells.
PMID:_12852827 [PubMed - in process]
Mol Pharmacol. 2003 Aug;64(2):308-15. c-Jun NH2-terminal kinase inhibitor anthra(1,9-cd)pyrazol-6(2H)-one reduces inducible nitric-oxide synthase expression by destabilizing mRNA in activated macrophages.
Lahti A, Jalonen U, Kankaanranta H, Moilanen E.
The Immunopharmacological Research Group, University of Tampere Medical School, Tampere, Finland.
In this study, we investigated the role of c-Jun NH2-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, in lipopolysaccharide (LPS)-stimulated inducible nitric-oxide synthase (iNOS) expression and nitric oxide (NO) production in J774 murine macrophages. Anthra(1,9-cd)pyrazol-6(2H)-one (SP600125), a pharmacological inhibitor of JNK, inhibited phosphorylation of c-Jun with an IC50 of 5 to 10 microM. At the same concentrations, SP600125 inhibited LPS-induced iNOS protein expression and NO production. SP600125 had no effect on the activation of nuclear factor kappaB, which is an important transcription factor for iNOS expression. SP600125 had no significant effect on iNOS mRNA levels if measured 4 h after LPS. In contrast, SP600125 reduced iNOS mRNA levels >90% when measured 8 h after LPS. These data suggest that SP600125 reduced iNOS mRNA stability, and this was confirmed in the mRNA degradation assay using actinomycin D, in which SP600125 reduced the iNOS mRNA half-life from 5 to 2 h. These results show that the JNK pathway is involved in the up-regulation of LPS-induced iNOS expression and NO production by a mechanism related to the stabilization of iNOS mRNA.
PMID:_12869635
Zhonghua Jie He He Hu Xi Za Zhi. 2003 Jun;26(6):358-61. [The inhibitory mechanism of nitri-oxide synthase gene transfection on hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells]
[Article in Chinese]
Zeng Q, Ran PX, Chen SC, Liu JS.
Guangzhou Institute of Respiratory Diseases, The First Affiliated Hospital, Guangzhou Medical College, Guangzhou 510120, China.
OBJECTIVE: The underlying mechanism by which nitric-oxide synthase (iNOS) gene transfer inhibits hypoxia-induced PASMCs proliferation remains unknown. The aim of this study is to investigate if iNOS gene transfer to PASMCs during hypoxia has any effect on cell cycle progression. METHODS: Using the cationic liposome mediation method, we transfected a recombinant pLNCX/Inos vector into rat PASMCs. The instantaneous transgenic expression and the function of the recombinant protein were detected. Cell cycle analysis was performed by flow cytometry and cell proliferation assay by [(3)H] thymidine incorporation. The proteins involved in cell cycle control (P27 and P21) were determined by RT-PCR and flow cytometry. RESULTS: iNOS expression was detected in the transfected PASMCs. NO(2)(-) levels were increased in iNOS-transfected cells as compared to the untransfected cells. Expression of iNOS in rat PASMCs under hypoxia resulted in a delay in inhibition of DNA synthesis and cell cycle progression. The incorporation of [(3)H] thymidine in iNOS-transfected group (15 145 +/- 1 514) dpm was significantly lower than those in the hypoxia group (18 011 +/- 2 521) dpm (P < 0.01). The G(0)/G(1) cell cycle arrest rate in the iNOS-transfected group (67.8%) was significantly higher than those in the hypoxia group (46.8%) (P < 0.01). The protein level of P27 was down-regulated by hypoxia but not in iNOS-transfected cells under hypoxia, and the level of the latter was similar to that under normoxia. CONCLUSIONS: Pre-transfer of iNOS gene to PASMCs under hypoxia inhibits cell proliferation via blocking P27 down-regulation, which is an important mechanism for the delay of cell cycle progression.
PMID:_12899770 [PubMed - in process]
Free Radic Biol Med. 2003 Aug 15;35(4):381-96. Nitric oxide and cGMP activate the Ras-MAP kinase pathway-stimulating protein tyrosine phosphorylation in rabbit aortic endothelial cells.
Department of Biochemistry, Fundacao Pro-Sangue Hemocentro de S. Paulo, Sao Paulo, Brazil.
The free radical nitric oxide is a very effective signal transducer, stimulating the enzyme guanylyl cyclase, the oncoprotein p21Ras, and protein tyrosine phosphorylation. In the present study using rabbit aortic endothelial cells (RAEC), it is demonstrated that the nitric-oxide-generating substances sodium nitroprusside and S-nitroso-N-acetylpenicillamine, and a stable analog of cyclic GMP, 8BrcGMP stimulate p21Ras activity. Tyrosine phosphorylation of cytosolic proteins was stimulated and intracellular production of cGMP was increased, indicating that the NO/cGMP-stimulated tyrosine phosphorylation-dependent signaling pathway is most likely associated with the activation of p21Ras. NO and cGMP-dependent activation of p21Ras result in binding of the oncoprotein to the Ras-binding domain of Raf-1 kinase. Incubation of RAEC with FPT II, a potent and selective inhibitor of p21Ras, prevented NO-dependent tyrosine phosphorylation. ODQ, a potent inhibitor of the soluble form of guanylyl cyclase, inhibited the signal as well. Conversely, the use of KT5823, a cGMP-dependent protein kinase (PKG) blocker, showed no effect on protein tyrosine phosphorylation. To further establish a role for p21Ras on the NO-stimulated tyrosine phosphorylation-signaling pathway, RAEC were constitutively transfected with a dominant negative mutant of p21Ras, N17Ras. NO and cGMP-stimulated tyrosine phosphorylation were prevented in N17Ras-expressing RAEC exposed to NO donors and 8BrcGMP. The above findings indicate that NO and cGMP stimulation of protein tyrosine phosphorylation requires the participation of fully functional p21Ras. ERK1/2 MAP kinases and their subsequent targets, the transcription factors, lie downstream to Ras, Raf-1 kinase, and MEK. Treatment of both RAEC and mock-transfected RAEC with NO resulted in phosphorylation and activation of ERK1/2. On the other hand, NO did not stimulate phosphorylation of ERK1/2 in N17Ras-expressing RAEC. In addition, PD98059, a MEK inhibitor, prevented overall tyrosine phosphorylation and phosphorylation of ERK1/2. Upstream to Ras ERK1/2 MAP kinases target the EGF receptor. Incubation of RAEC or mock-transfected RAEC with NO donors resulted in activation of the EGF receptor autophosphorylation. PD98059 effectively blocked this activation. EGF receptor autophosphorylation was insensitive to NO stimulation in N17Ras-expressing RAEC. It is concluded that NO and cGMP stimulate a signaling pathway involving p21Ras-Raf-1 kinase-MEK-ERK1/2. Activation of this signaling pathway is connected to NO-stimulated overall tyrosine phosphorylation that also involves the transactivation of the EGF receptor mediated by ERK1/2.
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