The highly related LIM factors, LMO1, LMO3 and LMO4, play different roles in the regulation of the pituitary glycoprotein hormone alpha-subunit (alpha GSU) 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.
Susa
Takao
T
Division of Life Science, Graduate School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kanagawa 214-8571, Japan.
Ishikawa
Akio
A
Cai
Li-yi
LY
Kato
Takako
T
Matsumoto
Kaori
K
Kitahara
Kousuke
K
Kurokawa
Rei
R
Ono
Tetsuo
T
Kato
Yukio
Y
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics* / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D060588:LIM Domain Proteins; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D010902:Pituitary Gland / Q000254:growth & development / Q000378:metabolism*; D010907:Pituitary Hormones / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D034421:Sus scrofa; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D015292:Glycoprotein Hormones, alpha Subunit; D018398:Homeodomain Proteins; D060588:LIM Domain Proteins; C065072:LMO1 protein, human; C502335:LMO3 protein, human; C114905:LMO4 protein, human; D015513:Oncogene Proteins; D010907:Pituitary Hormones; D014157:Transcription Factors