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Originally published In Press as doi:10.1074/jbc.M104162200 on July 10, 2001

J. Biol. Chem., Vol. 276, Issue 36, 34175-34181, September 7, 2001
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Cloning of a Novel Retinoid-inducible Serine Carboxypeptidase from Vascular Smooth Muscle Cells*

Jiyuan Chen, Jeffrey W. StrebDagger , Kathleen M. Maltby, Chad M. Kitchen, and Joseph M. Miano§

From the Center for Cardiovascular Research, University of Rochester Medical Center, Rochester, New York 14642

Retinoids block smooth muscle cell (SMC) proliferation and attenuate neointimal formation after vascular injury, presumably through retinoid receptor-mediated changes in gene expression. To identify target genes in SMC whose encoded proteins could contribute to such favorable biological effects, we performed a subtractive screen for retinoid-inducible genes in cultured SMC. Here, we report on the cloning and initial characterization of a novel retinoid-inducible serine carboxypeptidase (RISC). Expression of RISC is low in cultured SMC but progressively increases over a 5-day time-course treatment with all-trans-retinoic acid. A near full-length rat RISC cDNA was cloned and found to have a 452-amino acid open reading frame containing an amino-terminal signal sequence, followed by several conserved domains comprising the catalytic triad common to members of the serine carboxypeptidase family. In vitro transcription and translation experiments showed that the rat RISC cDNA generates a ~51-kDa protein. Confocal immunofluorescence microscopy of COS-7 cells transiently transfected with a RISC-His tag plasmid revealed cytosolic localization of the fusion protein. Western blotting studies using conditioned medium from transfected COS-7 cells suggest that RISC is a secreted protein. Tissue Northern blotting studies demonstrated robust expression of RISC in rat aorta, bladder, and kidney with much lower levels in all other tissues analyzed; high level RISC expression was also observed in human kidney. In situ hybridization verified the localization of RISC to medial SMC of the adult rat aorta. Interestingly, expression in kidney was restricted to proximal convoluted tubules; little or no expression was observed in glomerular cells, distal convoluted and collecting tubules, or medullary cells. Radiation hybrid mapping studies placed the rat RISC locus on chromosome 10q. These studies reveal a novel retinoid-inducible protease whose activity may be involved in vascular wall and kidney homeostasis.


* This work was supported in part by Scientist Development Grant 9730145N (to J. M. M.) and by Postdoctoral Fellowship Grant 0020233T from the American Heart Association (to J. C.).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 GenBankTM/EMBL Data Bank with accession number(s) AF330051 (rat RISC) and AF330052 (mouse RISC).

Dagger Recipient of National Institutes of Health Cardiovascular Research Training Grant 1T32HL07949.

§ To whom correspondence should be addressed: Center for Cardiovascular Research, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-273-1664; Fax: 716-273-1497; E-mail: joseph_miano@urmc.rochester.edu.


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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