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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4264-4276
Physical Interaction Between Retinoic Acid Receptor and Sp1:
Mechanism for Induction of Urokinase by Retinoic Acid
Yasuhiro Suzuki,
Jun Shimada,
Koichi Shudo,
Masatoshi Matsumura,
Massimo P. Crippa, and
Soichi Kojima
From the Laboratory of Molecular Cell Sciences, Tsukuba Life Science
Center, The Institute of Physical and Chemical Research (RIKEN),
Tsukuba, Ibaraki, Japan; the Institute of Applied Biochemistry,
University of Tsukuba, Tennoudai, Tsukuba, Ibaraki, Japan; the Faculty
of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; and the
Laboratory of Molecular Genetics, DIBIT-H. S. Raffaele, Milano, Italy.
Induction of urokinase plasminogen activator (uPA) by retinoic acid
(RA) is the initial event preceding certain subsequent biological
changes in vascular endothelial cells. We investigated the molecular
mechanism by which RA stimulates the expression of uPA, which lacks a
canonical RA receptor (RAR)-responsive element, in bovine and human
aortic endothelial cells. Upon stimulation with RA, mRNA levels of
RAR and  transiently increased in parallel with the induction of
uPA, and this increase was inhibited by cycloheximide. Results of
transient transfection of RAR/RXR cDNAs and experiments using specific
agonists and antagonists suggested that uPA induction is dependent upon
RAR (initially, RAR) with the help of RXR. Deletion analysis of
the uPA promoter suggested that RAR/RXR acts on GC box region within
the uPA promoter. This was further supported by inhibition of Sp1
binding to this region. Coimmunoprecipitation studies, glutathione
S-transferase pull-down experiment, and mammalian two-hybrid
assays suggested a physical interaction between RAR/RXR and Sp1.
Furthermore, gel shift studies showed that the binding of Sp1 to the
uPA GC box is significantly potentiated in the presence of RARs/RXRs.
Finally, Sp1 and RAR/RXR synergistically enhanced the transactivation
activity of the uPA promoter. These results suggest that (1) RA induces
RARs mainly via RAR and that (2) RAR/RXR physically and functionally
interact with Sp1, resulting in a potentiation of uPA transcription.
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