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

Abstract

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. DESIGN: 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. RESULTS AND CONCLUSION: 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.

A disputed evidence on obesity: comparison of the effects of Rcan2−/− and Rps6kb1−/− mutations on growth and body weight in C57BL/6J mice

“…In mice, two splice variants of Rcan2 that display distinct tissue-specific expression patterns have been identified (Mizuno et al, 2004): Rcan2-3 is expressed predominately in the brain, whereas Rcan2-1 is also expressed in other tissues, such as the heart and skeletal muscle. Recently, it was reported that loss of Rcan2 function in mice did not affect their linear growth (Bassett et al, 2012), but significantly ameliorated age-and dietinduced obesity by causing a reduction in food intake (Sun et al, 2011).…”

Spatial and Intracellular Distribution of the Endogenous Calcineurin-Inhibitory Proteins, ZAKI-4, in Mouse Brain

“…In the neonatal rat brain, expression of ZAKI-4 mRNA in cerebral cortex gradually increases with age but the increase is attenuated in hypothyroid pups [26]. Recently, we have also shown in the adult mouse brain that the expression of ZAKI-4 a mRNA is significantly decreased by hypothyroidism [19].…”

“…We have previously shown by Northern blot analysis that both ZAKI-4 a and b mRNAs are abundantly expressed in the brain [4,19]. The abundant expression of ZAKI-4 isoforms in brain is distinct among endogenous calcineurin inhibitors that contain an ISPPxSPP motif.…”

“…The rest of the amino acid sequences in the C terminus are identical among these isoforms. These ZAKI-4 isoforms are also expressed in mice and show a distinct tissue distribution [19]. Northern blot analysis of mRNAs extracted from mouse and human tissues revealed that the expression of ZAKI-4 a mRNA was limited in the brain, whereas the b isoform was detected not only in the brain, but also in other tissues such as heart and skeletal muscles [4,19].…”

“…These ZAKI-4 isoforms are also expressed in mice and show a distinct tissue distribution [19]. Northern blot analysis of mRNAs extracted from mouse and human tissues revealed that the expression of ZAKI-4 a mRNA was limited in the brain, whereas the b isoform was detected not only in the brain, but also in other tissues such as heart and skeletal muscles [4,19]. Since both ZAKI-4 a and b proteins contain the ISPPxSPP motif, which has been shown to be important for calcineurin inhibition [13], these isoforms seem to inhibit calcineurin activity and thus may play a role in the development and functions of the brain.…”