Abstract.We cloned the LIM-homeodomain protein LHX2 as a transcription factor for the porcine follicle-stimulating hormone β subunit gene (Fshβ) by the Yeast One-Hybrid Cloning System using the upstream region of -852/-746 bases (b) from the transcription start site, called Fd2, as a bait sequence. The reporter assay in LβT2 and CHO cells revealed the presence of an LHX2-responsive region other than Fd2. A potential LHX2 binding sequence was confirmed as AATTAAT containing a consensus homeodomain binding core sequence AATT by Systematic Evolution of Ligands by Exponential Enrichment analysis. DNase I footprinting demonstrated three AATTAAT sequences located at regions -835/-829, -818/-812 and -806/-800 b in the Fd2 region and 12 binding sites in the distal and proximal regions mostly containing an AATT-core sequence. RT-PCR analysis of Lhx2 expression during porcine fetal and postnatal pituitary development showed a gradual increase from fetal day (f) 40 to postnatal day (p) 8 followed by a slight decrease to p230, suggesting that LHX2 may play its role largely in the late fetal and postnatal periods. The analyses of Lhx2 expression in pituitary tumor-derived cell lines showed their expressions in cell lines including αT31, LβT2 and others. Since LHX2 was previously identified as a transcription factor for Cga and the in vitro experiments in the present study suggested that LHX2 regulated the expression of Fshβ, it is possible that LHX2 controls the synthesis of FSH at the transcription level. Key words: FSH, Gene regulation, Glycoprotein hormone, Lhx2, Pituitary (J. Reprod. Dev. 58: [147][148][149][150][151][152][153][154][155] 2012) F ollicle-stimulating hormone (FSH) is a member of the pituitary glycoprotein hormone family that includes both a luteinizing hormone (LH) and a thyroid-stimulating hormone (TSH). Each of these hormones shares a glycoprotein hormone common α subunit (αGSU) and contains a unique β subunit that confers biological specificity on its respective hormone function. The synthesis and secretion of FSH and LH are restricted to pituitary gonadotropes. Since the regulatory mechanisms of the three subunit genes that form gonadotropins are of special interest, several approaches have already achieved and provided a better understanding of the molecular mechanisms and regulatory factors governing the basal and cell-specific expression of the αGSU gene (Cga) and Lhβ [1]. On the other hand, our knowledge of the regulation of Fshβ expression remains limited to the information from extracellular signals. GnRH stimulation of FSH is mediated by the protein kinase C signaling cascade via AP-1 sites in the region of -120/-83 bases (b) from the transcription start site [2][3][4] and the distal region between -4152/-2878 and -2550/-1089 b of ovine Fshβ [5]. We previously observed that GnRH significantly stimulates the gene expression of c-Jun and c-Fos, components of AP-1 factor [6]. Multiple progesterone response elements (PREs) have been identified in the proximal region of ovine [7] and rat Fshβ [8]....
Recently, we have reported that a Prophet of Pit-1 homeodomain factor, Prop-1, is a novel transcription factor for the porcine follicle-stimulating hormone b subunit (FSHb) gene. This study subsequently aimed to examine the role of Prop-1 in the gene expression of two other porcine gonadotropin subunits, pituitary glycoprotein hormone a subunit (aGSU), and luteinizing hormone b subunit (LHb). A series of deletion mutants of the porcine aGSU (up to K1059 bp) and LHb (up to K1277 bp) promoters were constructed in the reporter vector, fused with the secreted alkaline phosphatase gene (pSEAP2-Basic). Transient transfection studies using GH3 cells were carried out to estimate the activation of the porcine aGSU and LHb promoters by Prop-1, which was found to activate the aGSU promoter of K1059/C12 bp up to 11 . 7-fold but not the LHb promoter. Electrophoretic mobility shift assay and DNase I footprinting analysis revealed that Prop-1 binds to six positions, K1038/K1026, K942/K928, K495/K479, K338/K326, K153/K146, and K131/K124 bp, that comprise the A/T cluster. Oligonucleotides of six Prop-1 binding sites were directly connected to the minimum promoter of aGSU, fused in the pSEAP2-Basic vector, followed by transfecting GH3 cells to determine the cis-acting activity. Finally, we concluded that at least five Prop-1 binding sites are the cis-acting elements for aGSU gene expression. The present results revealed a notable feature of the proximal region, where three Prop-1-binding sites are close to and/or overlap the pituitary glycoprotein hormone basal element, GATA-binding element, and junctional regulatory element. To our knowledge, this is the first demonstration of the role of Prop-1 in the regulation of aGSU gene expression. These results, taken together with our previous finding that Prop-1 is a transcription factor for FSHb gene, confirm that Prop-1 modulates the synthesis of FSH at the transcriptional level. On the other hand, the defects of Prop-1 are known to cause dwarfism and combined pituitary hormone deficiency accompanying hypogonadism. Accordingly, the present observations provide a novel view to understand the hypogonadism caused by Prop-1 defects at the molecular level through the regulatory mechanism of aGSU and FSHb gene expressions.
Abstract. Gene expression of the porcine glycoprotein hormone α subunit (p-αGSU) was examined in LβT2 cells, which were established from the anterior pituitary lobe of the immortalized transgenic mouse and produce αGSU, and in CHO cells cloned from Chinese hamster ovaries. Expression of the reporter gene fused with p-αGSU gene upstream in LβT2 cells showed that the distal regions -540/-240 and -798/-541 are important for the activation of gene expression. In contrast, the transcriptional activity of the distal region of p-αGSU gene was repressed in CHO cells. The region -540/-240 contains an adequate enhancer, called pituitary glycoprotein hormone basal element, whereas the region -798/-541 has no distinguished element. Transfection of the expression vector containing cDNA of a pan-pituitary activator, Ptx1, whose putative binding sites are present scatted in the distal region of the p-αGSU gene, revealed unexpectedly that this factor significantly suppressed the expression of p-αGSU gene in LβT2 cells, indicating that Ptx1 is unrelated to the upregulation in the region -798/-541. Thus, this study demonstrated for the first time that the distal region -798/-541of the p-αGSU gene is indispensable for prominent expression of this gene in which an as yet unidentified factor may participate.
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.
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