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J Biol Chem, Vol. 275, Issue 19, 14139-14146, May 12, 2000
From the Laboratory of Environmental Molecular Physiology, School
of Life Science, Tokyo University of Pharmacy and Life Science,
1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Accumulated evidence indicates that hypoxia
activates collagen synthesis in tissues. To explore the molecular
mechanism of activation, we screened genes that are up-regulated or
down-regulated by hypoxia. Fibroblasts isolated from fetal rat lung
were cultured under hypoxia. Differential display technique showed that
the mRNA level of prolyl 4-hydroxylase (PH) The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF197928.
Hypoxic Induction of Prolyl 4-Hydroxylase
(I) in Cultured
Cells*
§,,
(I), an active
subunit that catalyzes the oxygen-dependent hydroxylation
of proline residue in procollagen, increased 2-3-fold after an 8-h
exposure to hypoxia. This elevated level was maintained over 40 h
and returned to the basal level after reoxygenation. The transcription
rate, protein level, and hydroxyproline content (an indicator of the
prolyl hydroxylation) were all elevated by hypoxic culture. Analysis of
the promotor region of PH(I) gene indicated that a motif similar to
hypoxia-responsive element (HRE) of hypoxia-inducible genes such as
erythropoietin, was identified within a 120-base pair sequence upstream
of the transcription start site. Luciferase reporter assay and
mutational analysis showed that a site similar to the HRE in this motif
is functionally essential to hypoxic response. Electrophoretic mobility
shift assay revealed that hypoxia-inducible factor-1 was stimulated and
bound to the PH(I) HRE upon hypoxic challenge. Our results indicate
that PH(I), an essential enzyme for collagen synthesis, is a target
gene for hypoxia-inducible factor-1.
*
This work was supported in part by a grant-in-aid for
scientific research from the Ministry of Education, Science, Sports and
Culture of Japan (to Y. T. and S. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
These authors contributed equally to the completion of this study.
§
To whom correspondence should be addressed. Tel.: 81-426-76-7015;
Fax: 81-426-76-6811; E-mail: yuji@ls.toyaku.ac.jp.
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