Binding of upstream stimulatory factor 1 to the E-box regulates the 4G/5G polymorphism-dependent plasminogen activator inhibitor 1 expression in mast cells.
BACKGROUND
Plasminogen activator inhibitor (PAI)-1 is a key regulator of the fibrinolytic system. PAI-1 levels are markedly elevated in the asthmatic airways. The 4G/5G polymorphism of the PAI-1 gene is associated with allergic asthma.


OBJECTIVE
To characterize the mechanisms of the 4G/5G-dependent PAI-1 expression in mast cells (MCs), a major source of PAI-1 and key effector cells in asthma.


METHODS
Transcription of PAI-1 was assessed by transiently transfecting human MC line (HMC-1) cells with the luciferase-tagged PAI-1 promoters containing the 4G or 5G allele (4G-PAI-1 or 5G-PAI-1 promoter). Upstream stimulatory factor (USF)-1 and the E-box interactions were studied by electrophoretic mobility shift assays and supershift assays. Expression of USF-1 was determined by Western blot analysis.


RESULTS
The 4G-PAI-1 promoter has higher promoter activity than the 5G-PAI-1 promoter in stimulated HMC-1 cells, and the E-box adjacent to the 4G/5G site (E-4G/5G) regulates the genotype-specific PAI-1 transcription. USF-1 binds to the E-4G with greater affinity than to the E-5G. USF-1 level is increased in HMC-1 cells after stimulation, and elevated USF-1 enhances PAI-1 transcription. Overexpression of wild-type USF-1 or dominant-negative USF remedies the 4G/5G-dependent PAI-1 transcription.


CONCLUSIONS
Binding of USF-1 to the E-4G/5G regulates the 4G/5G polymorphism-dependent PAI-1 expression in MCs.
Ma
Zhongcai
Z
Division of Allergy and Immunology, Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, CA 90502, USA.
Jhun
Bongsook
B
Jung
Sandy Y
SY
Oh
Chad K
CK
D000483:Alleles; D001249:Asthma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D024721:E-Box Elements / Q000235:genetics / Q000502:physiology*; D006150:Guanine Nucleotides / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008407:Mast Cells / Q000276:immunology / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D011110:Polymorphism, Genetic / Q000502:physiology*; D011485:Protein Binding / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics; D051720:Upstream Stimulatory Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D006150:Guanine Nucleotides; D017395:Plasminogen Activator Inhibitor 1; C051321:SERPINE1 protein, human; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors