Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone α subunit gene expression

Abstract

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.

Regulation of Porcine Pituitary Glycoprotein Hormone Alpha Subunit Gene with LIM-Homeobox Transcription Factor Lhx3

“…Recently, we identified Prop1, a paired-like homeodomain transcription factor crucial for early pituitary development and regulation of pituitary-specific transcription factor Pit1 [34], as a candidate novel regulator for Cga [5] as well as Fshb [35]. Table 2.…”

“…The binding reaction mixture included 100 fmol of FAM-labeled probe DNA and 100 ng of recombinant ΔLhx3b protein with 2000 ng poly (dI-dC) in 10 μl of binding buffer (10 mM Hepes buffer, pH 7.9, containing 0.4 mM MgCl2, 0.4 mM DTT, 50 mM NaCl and 4% glycerol) at 30 C for 30 min. EMSA was carried out by electrophoresis on a 4% polyacrylamide gel as described previously [5].…”

“…The mixture was subjected to phenol-chloroform extraction, DNA precipitation and analysis on an ABI PRISM 310 Genetic Analyzer as described previously [5].…”

“…It is expressed differently in gonadotrope cells producing FSH and LH and in thyrotrope cells producing TSH under cell-specific mechanisms [2]. Understanding the molecular basis of αGSU gene (Cga) regulation would be of scientific interest.We previously cloned and determined up to the -1059 bp upstream sequence of the porcine Cga [3] and demonstrated that transcription factors, Ptx1, Prop1 and Msx1, are potentially capable of modulating this gene [4][5][6]. Several studies have demonstrated that many cis-elements are important for the response of the extracellular signal and cell type-or tissue-specific expression of Cga [7,8].…”

“…The binding reaction mixture included 100 fmol of FAM-labeled probe DNA and 100 ng of recombinant ΔLhx3b protein with 2000 ng poly (dI-dC) in 10 μl of binding buffer (10 mM Hepes buffer, pH 7.9, containing 0.4 mM MgCl2, 0.4 mM DTT, 50 mM NaCl and 4% glycerol) at 30 C for 30 min. EMSA was carried out by electrophoresis on a 4% polyacrylamide gel as described previously [5].DNase I footprinting was performed for the binding mixture, which consisted of 5'-labeled DNA (100 fmol) and 400 ng recombinant ΔLhx3b protein with 250 ng poly (dI-dC) in binding buffer, at 30 C for 20 min, and this was followed by DNase I digestion with 0.1 or 0.2 units DNase I (RQ1 RNase-Free DNase, Promega, Madison, WI) for 15 min at 25 C. The reaction was stopped with 0.5 M EDTA to 65 mM. The mixture was subjected to phenol-chloroform extraction, DNA precipitation and analysis on an ABI PRISM 310 Genetic Analyzer as described previously [5].…”