Kousuke Kitahara

The highly related LIM factors, LMO1, LMO3 and LMO4, play different roles in the regulation of the pituitary glycoprotein hormone α-subunit (αGSU) gene.

LMO1, LMO3 and LMO4 were cloned from the adult porcine pituitary cDNA library. Amino acid sequences of porcine LMO1, LMO3 and LMO4 were highly conserved among mammalian species. Transfection assay of the pituitary-derived cell line L beta T2 was carried out using the pituitary alpha GSU (glycoprotein hormone alpha-subunit) promoter (-1059/+12 b) fused to pSEAP2-Basic vector as a reporter gene. The results demonstrated that, whereas LMO4 showed no apparent effect, alpha GSU promoter activity was markedly repressed by LMO1 but activated by LMO3, indicating the different roles of the three highly homologous proteins, LMO1, LMO3 and LMO4. Knockdown assay by LMO siRNAs (small interfering RNAs) confirmed the above results for LMO1 and LMO3, whereas that by LMO4 siRNA increased the expression, indicating different modes of action. RT-PCR (reverse transcription-PCR) for total RNAs of several cell lines showed that LMO1 and LMO4 mRNAs were present ubiquitously in all cell lines, except for LMO1 in L929 cells. In contrast, LMO3 mRNA was abundant only in L beta T4 and GH3 cells with only small amounts in L beta T2 and MtT/S cells, indicating the cell-type-specific function of this protein. Real-time analyses of porcine pituitary ontogeny revealed that the three LMO genes are expressed during the fetal period and decline immediately afterwards, followed by a remarkably low level of LMO3 and LMO4 after birth. RT-PCR of the porcine tissues examined showed ubiquitous expression of LMO4, whereas LMO1 and LMO3 are expressed tissue specifically. Thus the present study demonstrated that three highly related LIM cofactors, LMO1, LMO3 and LMO4, have different effects on alpha GSU gene expression in the pituitary glands.

Intracellular localization of porcine single‐strand binding protein 2

We have previously cloned cofactor CLIM2 (Ldb1/NL1) as a binding protein for LIM homeodomain transcription factor and now seek a protein interacting with CLIM2. Ultimately, SSBP2 was cloned as CLIM2 binding protein from the adult porcine pituitary cDNA library by the Yeast Two‐Hybrid System. The amino acid sequence of porcine SSBP2 shows a high identity (99%) with those of other mammalian species, man, and mouse. Using fluorescence protein‐fused SSBP2 and its deletion mutants, we observed that SSBP2 overexpressed in CHO cells predominantly localizes in mitochondria. Expression of mutant SSBP2s demonstrated that the first 241 amino acid residues are responsive for the mitochondrial localization. When CLIM2 vector was co‐transfected, SSBP2 changed its location to nuclei. The similar translocation was also observed when CLIM2 vector was transfected 17 h after the transfection of SSBP2 vector. The first 120 residues of SSBP2 are responsible for the nuclear localization by guidance with CLIM2. RT‐PCR demonstrated that SSBP2 was expressed in the porcine pituitary from fetal 40 days to postnatal 230 days in both genders and in the variety of pituitary and non‐pituitary tumor cell lines, indicating that SSBP2 is present ubiquitously and plays a universal function during fetal and postnatal pituitary development. J. Cell. Biochem. 106: 912–919, 2009.