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Originally published In Press as doi:10.1074/jbc.M301477200 on February 24, 2003

J. Biol. Chem., Vol. 278, Issue 19, 17084-17092, May 9, 2003
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An Inverse Correlation between Expression of a Preprocathepsin B-related Protein with Cysteine-rich Sequences and Steroid 11beta -Hydroxylase in Adrenocortical Cells*

Kuniaki MukaiDagger §, Fumiko MitaniDagger , Hideko NagasawaDagger , Reiko SuzukiDagger , Tsuneharu SuzukiDagger ||, Makoto SuematsuDagger , and Yuzuru IshimuraDagger **

From the Dagger  Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 and || Minophagen Pharmaceutical Co., 2-2-3 Komatsubara, Zama, Kanagawa 228-0002, Japan

A cDNA encoding a secretory protein hitherto unknown was cloned from mouse adrenocortical cells by subtractive hybridization between the cells without and with expressing steroid 11beta -hydroxylase (Cyp11b-1), a marker for the functional differentiation of cells in the zonae fasciculata reticularis (zFR). The deduced protein consisting of 466 amino acids contained a secretory signal, epidermal growth factor-like repeats, and a proteolytically inactive cathepsin B-related sequence. The amino acid sequence was 89% identical with that of human tubulointerstitial nephritis antigen-related protein. Among the mouse organs examined, adrenal glands prominently expressed its mRNA. The mRNA and its encoded protein were detected in the outer adrenocortical zones that do not express Cyp11b-1, i.e. the zona glomerulosa and the undifferentiated cell zone, while being undetectable in zFR that express Cyp11b-1. The new protein was designated as adrenocortical zonation factor 1 (AZ-1). Clonal lines with different levels of AZ-1 expression were established from Y-1 adrenocortical cells that originally express Cyp11b-1 but little AZ-1. Analyses of the clonal lines revealed that Cyp11b-1 is detected in the clonal lines maintaining little AZ-1 expression and becomes undetectable in those expressing AZ-1. On the other hand, irrespective of the AZ-1 expression, all clones expressed cholesterol side-chain cleavage enzyme, which occurs throughout the cortical zones. These results demonstrated that adrenocortical cells expressing AZ-1 do not express Cyp11b-1, whereas those with little AZ-1 express this zFR marker in vitro and in vivo, implying a putative role of AZ-1 in determining the zonal differentiation of adrenocortical cells.

* This work was supported in part by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science, by a national grant-in-aid for the Establishment of High-Tech Research Center in a Private University, and by grants from the Mitsubishi Foundation, the Uehara Memorial Foundation, the Ichiro Kanehara Foundation, and Keio University.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.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB050626.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel.: 81-3-5363-3752; Fax: 81-3-3358-8138; E-mail: mukaik@sc.itc.keio.ac.jp.

Present address: Dept. of Biological Science and Technology, Faculty of Engineering, the University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan.

** Present address: Dept. of Biochemistry, the University of Texas Health Science Center, San Antonio, TX 78229-3900.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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