Isx Participates in the Maintenance of Vitamin A Metabolism by Regulation of β-Carotene 15,15′-Monooxygenase (Bcmo1) Expression

Yusuke Seino,Takashi Miki,Hiroshi Kiyonari,Takaya Abe,Wakako Fujimoto,Keita Kimura,Ayako Takeuchi,Yoshihisa Takahashi,Yutaka Oiso,Toshihiko Iwanaga,Susumu Seino

doi:10.1074/jbc.m707928200

Abstract

Isx (intestine specific homeobox) is an intestine-specific transcription factor. To elucidate its physiological function, we generated Isx-deficient mice by knocking in the beta-galactosidase gene (LacZ) in the Isx locus (IsxLacZ/LacZ mice). LacZ staining of heterozygous (IsxLacZ/+) mice revealed that Isx was expressed abundantly in intestinal epithelial cells from duodenum to proximal colon. Quantitative mRNA expression profiling of duodenum and jejunum showed that beta-carotene 15,15'-monooxygenase (EC1.14.99.36 Bcmo1) and the class B type I scavenger receptor, which are involved in vitamin A synthesis and carotenoid uptake, respectively, were drastically increased in IsxLacZ/LacZ mice. Although mild vitamin A deficiency decreased Isx expression in duodenum of wild-type (Isx+/+) mice, severe vitamin A deficiency decreased Isx mRNA expression in both duodenum and jejunum of Isx+/+ mice. On the other hand, vitamin A deficiency increased Bcmo1 expression in both duodenum and jejunum of Isx+/+ mice. However, Bcmo1 expression was not increased in duodenum of IsxLacZ/LacZ mice by mild vitamin A deficiency. These data suggest that Isx participates in the maintenance of vitamin A metabolism by regulating Bcmo1 expression in the intestine.

Highlights

The epithelium of the small intestine comprises four types of cells: absorptive epithelial cells that absorb nutrients in the intestine, goblet cells that secrete mucus, Paneth cells that secrete antibacterial lysozyme, and gut endocrine cells that secrete various intestinal hormones [1]
To investigate expression of Isx in the crypt-villus axis of the intestine, the adult intestinal tract was subjected to in situ hybridization. mRNA expression of Isx in the small intestine was detected from the upper crypt to the villus along the axis, but in each villus, expression weakened toward the tip (Fig. 1C)
Microarray analysis and subsequent real time Reverse Transcriptase (RT)-PCR for HD transcription factors play critical roles in cell differentiation, cell proliferation, and organogenesis

Summary

EXPERIMENTAL PROCEDURES

Generation of IsxLacZ/LacZ Mice and Transgenic Mice Overexpressing Isx—Isx knock-out (IsxLacZ/LacZ) mice (accession number CDB0475K, Center for Developmental Biology, RIKEN, Kobe, Japan) were generated by replacing the amino acid coding sequences in exon 1 of Isx with LacZ as described (supplemental Fig. S1). In Situ Hybridization—Two nonoverlapping antisense oligonucleotide probes (45-mer in length) were designed for each mRNA of mouse Isx, Bcmo, and SR-BI. The antisense probes used were complementary to the following sequences: 631– 675 and 1031–1075 of mouse Isx (GenBankTM accession number AB219123); 427– 471 and 1520 –1564 of mouse Bcmo (GenBankTM accession number NM_021486); and 329 –373 and 953–997 of mouse SR-BI (GenBankTM accession number NM_016741). Southern Blot Analysis and Northern Blot Analysis—Southern blot analysis of mouse tail genomic DNA and Northern blot analysis of the tissues were performed by standardized procedures. Isxϩ/ϩ (n ϭ 4 –9) and IsxLacZ/LacZ (n ϭ 4 – 8) adult mice were fed a VAS diet for more than 4 weeks and placed on a diet with marginal or sufficient vitamin A content for 18 days. A probability level of p Ͻ 0.05 was considered statistically significant

RESULTS

DISCUSSION

A Mild vitamin A deficiency