[Effect of L-arginine on human coagulant factor VIII gene expression].
OBJECTIVE
To investigate the effect of L-arginine on expression of human FVIII gene.


METHODS
Plasmid pcDNA6/V5-HisA-BDDhF VIII containing B domain deleted human coagulant factor VIII cDNA (BDDhF VIII cDNA) was constructed and transfected into human umbilical vein endothelial cells (HUVEC). After 72 h incubation with L-arginine (final concentration was 10 mmol/L) , the supernatant was collected for determining the antigen and clotting activity of human FVIII (FVIII: Ag and FVIII: C ) with ELISA and one stage clotting assay respectively. HUVECs were harvested for detecting human FVIII mRNA by Northern blot analysis. The five functional domains of BDDhFVIII cDNA including A1, A2, A3, C1 and C2 were amplified with PCR and inserted into pcDNA6/V5-HisA to construct the expression plasmids pcDNA6/V5-Hi-sA-BDDhFVIII-A1, pcDNA6/V5-HisA-BDDhFVIII-A2, pcDNA6/V5-HisA-BDDhFVIII-A3, pcDNA6/V5-HisA-BDDhFVIII-C1 and pcDNA6/V5-HisA-BDDhFVIII-C2, respectively. HUVEC were transfected with the five plasmids respectively and incubated with L-arginine (at the final concentration of 10 mmol/L) for 72 h. Nucleoli were then isolated and underwent run-on assay.


RESULTS
After 24 h incubation with L-arginine, FVIII: Ag and FVIII: C were increased markedly in the supernatant of HUVEC [FVIII: Ag was (146.08 +/- 4.78) ng/ ml, and FVIII: C (0.752 +/- 0.009) U/ml/10(6) cells x 24 h, while in control supernatant without L-arginine, FVIII: Ag was (34.66 +/- 3.98) ng/ml, and FVIII: C (0.171 +/- 0.006) U/ml/10(6) cell x 24 h, P < 0.01]. Northern blot analysis indicated that, after adding L-arginine, the transcription of human FVIII mRNA was intensified remarkably in HUVEC transfected with pcDNA6/V5-HisA-BDDhFVIII, but no any transcription in those transfected with pcDNA6/V5-HisA. Run-on assay demonstrated that with L-arginine induction, A1 and A2 domains transcription was increased obviously, while no change in A3, C1 and C2 domains transcription.


CONCLUSIONS
L-arginine increases expression of human FVIII gene in HUVEC through enhancing its transcription, particularly, domain A1 and A2 within FVIII gene.
Yin
Jun
J
Department of Hematology, the Second Hospital Affiliated to Shantou University Medical College, Shantou, Guangdong Province 515041, China.
Shi
Gang-Gang
GG
Zheng
Qing-Tang
QT
Zeng
Yang
Y
Yao
Guan-Feng
GF
Lu
Pei-Wen
PW
Wang
Xue-Feng
XF
Wang
Hongli
H
D001120:Arginine / Q000494:pharmacology*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D042783:Endothelial Cells / Q000378:metabolism; D005169:Factor VIII / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D018076:DNA, Complementary; C078147:F8 protein, human; D005169:Factor VIII; D001120:Arginine