Genomic organization of mouse ZAKI-4 gene that encodes ZAKI-4 alpha and beta isoforms, endogenous calcineurin inhibitors, and changes in the expression of these isoforms by thyroid hormone in adult mouse brain and heart.
OBJECTIVE
ZAKI-4 was identified as a thyroid hormone-responsive gene in cultured human fibroblasts. A single ZAKI-4 gene encodes two isoforms, ZAKI-4 alpha and beta, both inhibiting calcineurin activity. ZAKI-4 alpha and beta differ at their N termini, and show distinct distribution profiles in human tissues. The aim of this study was to elucidate the organization of the mouse ZAKI-4 gene and to determine the effect of thyroid hormone on the expression of ZAKI-4 isoforms in vivo.


METHODS
We cloned mouse homologues of human ZAKI-4 alpha and beta cDNA. Fluorescence in situ hybridization and bioinformatics analysis were employed to determine the gene organization. The effect of thyroid hormone on the expression of ZAKI-4 isoforms in mouse brain and heart was also studied.


METHODS
Total RNA extracted from mouse cerebellum was used to clone ZAKI-4 alpha and beta cDNAs by RT-PCR followed by rapid amplification of cDNA ends. Mice were rendered hypothyroid by feeding a low iodine diet supplemented with propylthiouracil for 2 weeks. In one group (hyperthyroid) L-T(3) was injected i.p. for the last 4 days whereas another group (hypothyroid) received vehicle only. Non-treated mice were controls.


CONCLUSIONS
Mouse ZAKI-4 alpha and beta cDNAs were highly homologous to the human isoforms. The gene was mapped on chromosome 17qC, syntenic to human chromosome 6 where the human ZAKI-4 gene is located. As observed in human, ZAKI-4 alpha mRNA was expressed only in brain whereas beta mRNA was distributed in other tissues as well, such as heart and skeletal muscle. ZAKI-4 alpha mRNA was lower in the cerebral cortex of hypothyroid mice. Injection of L-T(3) caused an increase in ZAKI-4 beta mRNA in heart; however, expression of neither ZAKI-4 alpha nor beta mRNA was influenced by thyroid status in other tissues. These results indicate that expression of ZAKI-4 alpha and beta isoforms is regulated by thyroid hormone in vivo, and the regulation is isoform- and tissue-specific.
Mizuno
Yutaka
Y
Department of Endocrinology and Transplantation, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan.
Kanou
Yasuhiko
Y
Rogatcheva
Margarita
M
Imai
Tsuneo
T
Refetoff
Samuel
S
Seo
Hisao
H
Murata
Yoshiharu
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism / Q000502:physiology; D065095:Calcineurin Inhibitors; D002874:Chromosome Mapping; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D007037:Hypothyroidism / Q000235:genetics* / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics*; D009206:Myocardium / Q000378:metabolism; D020033:Protein Isoforms; D011506:Proteins; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D014018:Tissue Distribution; D014284:Triiodothyronine / Q000494:pharmacology*
D065095:Calcineurin Inhibitors; C486303:Dscr1l1 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D020033:Protein Isoforms; D011506:Proteins; D012333:RNA, Messenger; D014284:Triiodothyronine